PGC-1β, a novel PGC-1 homologue and uses therefor

ABSTRACT

The invention provides isolated nucleic acid molecules, designated PGC-1β nucleic acid molecules, which encode novel PGC-1 related coactivator molecules. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PGC-1β nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a PGC-1β gene has been introduced or disrupted. The invention still further provides isolated PGC-1β proteins, fusion proteins, antigenic peptides and anti-PGC-1β antibodies. Diagnostic and therapeutic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No. 10/290,544filed on Nov. 8, 2002, now U.S. Pat. No. 7,091,006 which claims priorityto U.S. Provisional Application No. 60/338,126 filed on Nov. 9, 2001;each application is incorporated herein in its entirety by reference.

GOVERNMENT SUPPORT

Work described herein was supported under grants R37DK31405 and DK54477awarded by the National Institutes of Health. The U.S. government mayhave certain rights in this invention.

BACKGROUND OF THE INVENTION

The transcriptional function of many nuclear receptors (NRs) isregulated by ligand-dependent recruitment of coactivators to thecarboxyl-terminal ligand-binding domain (Aranda, A., and Pascual, A.(2001) Physiol. Rev. 81:1269-1304; Rosenfeld, M. G. and Glass, C. K.(2001) J. Biol. Chem. 276:36865-36868). A number of coactivators,including the p160 family, p300/CBP and P/CAF, contain intrinsic histoneacetyl transferase activity and regulate transcription by modulatinghistone acetylation (Aranda and Pascual (2001) supra). Othercoactivators, consisting of heterogeneous proteins with little sequencehomology, modulate transcription by acting as protein docking interfacesthat recruit histone acetyl transferase-containing complexes orassociate with basal transcription factors such as RNA polymerase IIholoenzyme (Freedman, L. P. (1999) Cell 97:5-8). The interaction betweenNRs and many coactivators requires a conserved LXXLL motif (L is leucineand X is any amino acid), which is believed to form hydrophobic contactswith the receptors (Nolte, R. T. et al. (1998) Nature 395:137-143;Westin, S. et al. (1998) Nature 395:199-202).

PGC-1 was initially identified as a PPARγ-interacting protein from abrown adipose tissue (BAT) library and was subsequently found toassociate with an array of NRs and transcription factors (Puigserver, P.et al. (1998) Cell 92:829-839; Wu, Z. et al. (1999) Cell 98:115-124;Vega, R. B. et al. (2000) Mol. Cell. Biol. 20:1868-1876; Michael, L. F.et al. (2001) Proc. Natl. Acad. Sci. USA 98:3820-3825). Importantly,PGC-1 has been shown to coordinately regulate the program ofmitochondrial biogenesis and adaptive thermogenesis in BAT and skeletalmuscle, mainly through the coactivation of PPARs and nuclear respiratoryfactor 1 (NRF1), a nuclear transcription factor that regulates theexpression of many mitochondrial genes (Puigserver et al. (1998) supra;Wu et al. (1999) supra). In transgenic mice, PGC-1 increasesmitochondrial biogenesis and β-oxidation of fatty acids in the heart,likely through augmentation of PPARα and NRF1 transcriptional activity(Lehman, J. J. et al. (2000) J. Clin. Invest. 106:847-856). Recently,PGC-1 expression was found to be elevated in fasted liver and severalmodels of type-1 and type-2 diabetes; in addition, PGC-1 can directlycontrol the activation of hepatic gluconeogenesis (Yoon, J. C. et al.(2001) Nature 413:131-138; Herzig, S. et al. (2001) Nature 413:179-183).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel members of the family of PGC-1 molecules, referred to herein asPGC-1β nucleic acid and protein molecules (e.g., human and mousePGC-1β). The present invention is further based, at least in part, onthe discovery that PGC-1β is upregulated during brown fatdetermination/differentiation, but not during cold exposure. The presentinvention is further based, at least in part, on the discovery thatPGC-1β expression is upregulated in the liver during fasting. Thepresent invention is further based, at least in part, on the discoverythat PGC-1β induces mitochondrial biogenesis and fatty acid oxidationgene expression in the liver and cultured murine myotubes. The presentinvention is further based, at least in part, on the discovery thatPGC-1β is highly expressed in brown adipose tissue (BAT)and the heart(tissues that contain high levels of mitochondria and substrateoxidation)and on the discovery that PGC-1β is a potent coactivator ofNRF1. The present invention is still further based, at least in part, onthe discovery that host cell factor (HCF), a cellular protein that isinvolved in herpes simplex virus (HSV) infection and cell cycleregulation (Wilson, A. C. et al. (1993) Cell 74:115-125; Wilson, A. C.et al. (1997) Mol. Cell. Biol. 17:6139-6146), is a binding partner thatupregulates the transcriptional activity of both the originallyidentified PGC-1, hereinafter referred to as PGC-1α, and PGC-1β. Thepresent invention is further based, at least in part, on the discoverythat both PGC-1α and PGC-1β induce mitochondrial gene expression inneuroblastoma cells suggesting an important role in neurologicaldisorders. The present invention is yet further based, at least in part,on the discovery that both PGC-1α, and PGC-1β induce the expression ofenzymes involved in free radical metabolism such as superoxide dismutase(Mn-SOD) and glutathione peroxidase (GPx), suggesting an important rolein the cellular defense against free radical damage.

The PGC-1β nucleic acid and protein molecules of the present inventionare useful as modulating agents in regulating a variety of cellularprocesses, e.g., cellular determination and/or differentiation (e.g.,brown adipose determination and/or differentiation), cellularmetabolism, fatty acid oxidation, mitochondrial function and/orrespiration, cellular signaling, and/or cellular proliferation.Accordingly, in one aspect, this invention provides isolated nucleicacid molecules encoding PGC-1β proteins or biologically active portionsthereof, as well as nucleic acid fragments suitable as primers orhybridization probes for the detection of PGC-1β-encoding nucleic acids.

In one embodiment, a PGC-1β nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, 99.99% or more identical to the nucleotide sequence(e.g., to the entire length of the nucleotide sequence) shown in SEQ IDNO:1, 3, 4, or 6, or a complement thereof.

In a preferred embodiment, the isolated nucleic acid molecule includesthe nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, or acomplement thereof. In another preferred embodiment, the isolatednucleic acid molecule includes nucleotides 1-660 of SEQ ID NO:3. Inanother preferred embodiment, the isolated nucleic acid moleculecomprises nucleotides 1-1140 of SEQ ID NO:3. In another preferredembodiment, the nucleic acid molecule consists of the nucleotidesequence shown in SEQ ID NO:1, 3, 4, or 6.

In another embodiment, a PGC-1β nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently identical to the amino acid sequence of SEQ ID NO:2 or 5.In a preferred embodiment, a PGC-1β nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequence atleast 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90%,95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%,99.7%, 99.8%, 99.9%, 99.99% or more identical to the entire length ofthe amino acid sequence of SEQ ID NO:2 or 5.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of human PGC-1β. In another preferredembodiment, an isolated nucleic acid molecule encodes the amino acidsequence of mouse PGC-1β. In yet another preferred embodiment, thenucleic acid molecule includes a nucleotide sequence encoding a proteinhaving the amino acid sequence of SEQ ID NO:2 or 5. In yet anotherpreferred embodiment, the nucleic acid molecule is at least 50, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 457, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300,1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900,1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500,2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100,3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600 or morenucleotides in length. In a further preferred embodiment, the nucleicacid molecule is at least 50, 75, 100, 125, 150, 175, 200, 250, 300,350, 400, 450, 457, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950,1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550,1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150,2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550, 2600, 2650, 2700, 2750,2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150, 3200, 3250, 3300, 3350,3400, 3450, 3500, 3550, 3600 or more nucleotides in length and encodes aprotein having a PGC-1β activity (as described herein).

Another embodiment of the invention features nucleic acid molecules,preferably PGC-1β nucleic acid molecules, which specifically detectPGC-1β nucleic acid molecules relative to nucleic acid moleculesencoding non-PGC-1β proteins. For example, in one embodiment, such anucleic acid molecule is at least 20, 30, 40, 50, 75, 100, 125, 150,175, 200, 250, 300, 350, 400, 450, 457, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350,1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950,2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450, 2500, 2550,2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150,3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600 or more nucleotidesin length and hybridizes under stringent conditions to a nucleic acidmolecule comprising the nucleotide sequence shown in SEQ ID NO:1 or 4,or a complement thereof.

In preferred embodiments, the nucleic acid molecules are at least 15(e.g., 15 contiguous) nucleotides in length and hybridize understringent conditions to the nucleotide molecules set forth in SEQ IDNO:1 or 4.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2 or 5, wherein the nucleic acidmolecule hybridizes to a complement of a nucleic acid moleculecomprising SEQ ID NO:1, 3, 4, or 6, respectively, under stringentconditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a PGC-1β nucleic acid molecule, e.g., thecoding strand of a PGC-1β nucleic acid molecule.

Another aspect of the invention provides a vector comprising a PGC-1βnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. In yetanother embodiment, the invention provides a host cell containing anucleic acid molecule of the invention. The invention also provides amethod for producing a protein, preferably a PGC-1β protein, byculturing in a suitable medium, a host cell, e.g., a mammalian host cellsuch as a non-human mammalian cell, of the invention containing arecombinant expression vector, such that the protein is produced.

Another aspect of this invention features isolated or recombinant PGC-1βproteins and polypeptides. In one embodiment, an isolated PGC-1β proteinincludes at least one or more of the following domains: an LXXLL motif,an RRM, an AD, an HBM, and/or a glutamic/aspartic acid rich acidicdomain.

In a preferred embodiment, a PGC-1β protein includes at least one ormore of the following domains: an LXXLL motif, an RRM, an AD, an HBM,and/or a glutamic/aspartic acid rich acidic domain, and has an aminoacid sequence at least about 50%, 55%, 60%, 65%, 67%, 68%, 70%, 71%,72%, 73%, 74%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%,99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or moreidentical to the amino acid sequence of SEQ ID NO:2 or 5.

In another preferred embodiment, a PGC-1β protein includes at least oneor more of the following domains: an LXXLL motif, an RRM, an AD, an HBM,and/or a glutamic/aspartic acid rich acidic domain, and has a PGC-1βactivity (as described herein).

In yet another preferred embodiment, a PGC-1β protein includes at leastone or more of the following domains: an LXXLL motif, an RRM, an AD, anHBM, and/or a glutamic/aspartic acid rich acidic domain, and is encodedby a nucleic acid molecule having a nucleotide sequence which hybridizesunder stringent hybridization conditions to a complement of a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1, 3, 4,or 6.

In another embodiment, the invention features fragments of the proteinhaving the amino acid sequence of SEQ ID NO:2 or 5, wherein the fragmentcomprises at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125,150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,800, 850, 900, 950, or 1000 amino acids (e.g., contiguous amino acids)of the amino acid sequence of SEQ ID NO:2 or 5. In another embodiment, aPGC-1β protein has the amino acid sequence of SEQ ID NO:2 or 5.

In another embodiment, the invention features a PGC-1β protein which isencoded by a nucleic acid molecule consisting of a nucleotide sequenceat least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 80%,85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%,99.6%, 99.7%, 99.8%, 99.9%, 99.99% or more identical to a nucleotidesequence of SEQ ID NO:1, 3, 4, or 6, or a complement thereof. Thisinvention further features a PGC-1β protein which is encoded by anucleic acid molecule consisting of a nucleotide sequence whichhybridizes under stringent hybridization conditions to a complement of anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,3, 4, or 6, or a complement thereof.

The proteins of the present invention or portions thereof, e.g.,biologically active portions thereof, can be operatively linked to anon-PGC-1β polypeptide (e.g., heterologous amino acid sequences) to formfusion proteins. The invention further features antibodies, such asmonoclonal or polyclonal antibodies, that specifically bind proteins ofthe invention, preferably PGC-1β proteins. In addition, the PGC-1βproteins or biologically active portions thereof can be incorporatedinto pharmaceutical compositions, which optionally includepharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a PGC-1β nucleic acid molecule, protein, or polypeptidein a biological sample by contacting the biological sample with an agentcapable of detecting a PGC-1β nucleic acid molecule, protein, orpolypeptide such that the presence of a PGC-1β nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of PGC-1β activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofPGC-1β activity such that the presence of PGC-1β activity is detected inthe biological sample.

In another aspect, the invention provides a method for modulating PGC-1βactivity comprising contacting a cell capable of expressing PGC-1β withan agent that modulates PGC-1β activity such that PGC-1β activity in thecell is modulated. In one embodiment, the agent inhibits PGC-1βactivity. In another embodiment, the agent stimulates PGC-1β activity.In one embodiment, the agent is an antibody that specifically binds to aPGC-1β protein. In another embodiment, the agent modulates expression ofPGC-1β by modulating transcription of a PGC-1β gene or translation of aPGC-1β mRNA. In yet another embodiment, the agent is a nucleic acidmolecule having a nucleotide sequence that is antisense to the codingstrand of a PGC-1β mRNA or a PGC-1β gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant or unwantedPGC-1β protein or nucleic acid expression or activity by administeringan agent which is a PGC-1β modulator to the subject. In one embodiment,the PGC-1β modulator is a PGC-1β protein. In another embodiment thePGC-1β modulator is a PGC-1β nucleic acid molecule. In yet anotherembodiment, the PGC-1β modulator is a peptide, peptidomimetic, or othersmall molecule. In a preferred embodiment, the disorder characterized byaberrant or unwanted PGC-1β protein or nucleic acid expression is aPGC-1β-associated disorder, e.g., a metabolic disorder or a neurologicaldisorder, as described herein. In another preferred embodiment, thedisorder is characterized by free radical damage.

The present invention also provides diagnostic assays for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aPGC-1β protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a PGC-1β protein, wherein a wild-typeform of the gene encodes a protein with a PGC-1β activity.

In another aspect the invention provides methods for identifying acompound that binds to or modulates the activity of a PGC-1β protein, byproviding an indicator composition comprising a PGC-1β protein havingPGC-1β activity, contacting the indicator composition with a testcompound, and determining the effect of the test compound on PGC-1βactivity in the indicator composition to identify a compound thatmodulates the activity of a PGC-1β protein.

In other embodiments, the invention provides methods for identifying asubject having a metabolic disorder, or at risk for developing ametabolic disorder; methods for identifying a compound capable oftreating a metabolic disorder characterized by aberrant PGC-1 nucleicacid expression or PGC-1 polypeptide activity; and methods for treatinga subject having a metabolic disorder characterized by aberrant PGC-1polypeptide activity or aberrant PGC-1 nucleic acid expression.

In yet other embodiments, the invention provides methods for identifyinga subject having a neurological disorder, or at risk for developing aneurological disorder; methods for identifying a compound capable oftreating a neurological disorder characterized by aberrant PGC-1 nucleicacid expression or PGC-1 polypeptide activity; and methods for treatinga subject having a neurological disorder characterized by aberrant PGC-1polypeptide activity or aberrant PGC-1 nucleic acid expression.

In yet further embodiments, the invention provides methods foridentifying a subject having a disorder characterized by free radicaldamage to cells, or at risk for developing a disorder characterized byfree radical damage to cells; methods for identifying a compound capableof treating a disorder characterized by free radical damage to cellscharacterized by aberrant PGC-1 nucleic acid expression or PGC-1polypeptide activity; and methods for treating a subject having such adisorder characterized by aberrant PGC-1 polypeptide activity oraberrant PGC-1 nucleic acid expression.

In another embodiment, the invention provides a method for identifying acompound which modulates the interaction of a PGC-1α protein with an HCFprotein comprising contacting, in the presence of the compound, thePGC-1α protein and the HCF protein under conditions which allow bindingof the HCF protein to the PGC-1α protein to form a complex; anddetecting the formation of a complex of the PGC-1α protein and the HCFprotein in which the ability of the compound to modulate the interactionbetween the PGC-1 protein and the HCF protein is indicated by a changein complex formation as compared to the complex formed (e.g., structureand/or amount of complex formed) in the absence of the compound.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B depict the cDNA sequence of murine PGC-1β. The nucleotidesequence corresponds to nucleic acids 1 to 3664 of SEQ ID NO:1. Thetranslation start codon and the stop codon are underlined.

FIG. 2 depicts the predicted protein sequence of murine PGC-1β. Theamino acid sequence corresponds to residues 1 to 1014 of SEQ ID NO:2.The three LXXLL motifs are underlined, the RRM (RNA binding motif) isshown in bold, and the HBM (host cell binding factor motif) is boxed.

FIGS. 3A-3B depict the cDNA sequence of human PGC-1β. The nucleotidesequence corresponds to nucleic acids 1 to 3030 of SEQ ID NO:4. Thetranslation start codon and the stop codon are underlined.

FIG. 4 depicts the predicted protein sequence of human PGC-1β. The aminoacid sequence corresponds to residues 1 to 1009 of SEQ ID NO:5. The twoLXXLL motifs are underlined, the RRM (RNA binding motif) is shown inbold, and the HBM (host cell binding factor motif) is boxed.

FIG. 5 depicts a schematic diagram of an alignment between murinePGC-1α, PGC-1β and PRC. The percent identity of the regions of eachprotein to PGC-1β is indicated. Conserved domains and/or motifs includethe activation domain (AD), LXXLL and HBM motifs, the RS domain, and theRRM motif. The HBM motif is conserved in all three PGC-1 relatedproteins.

FIG. 6 depicts a schematic diagram of the genomic structure andchromosomal localization of the murine and human PGC-1β genes.

FIGS. 7A-7D depict the results of transcriptional analysis of thecoactivation of nuclear receptors by murine PGC-1β. COS cells werecotransfected with vectors expressing GR (FIG. 7A), HNF4α (FIG. 7B),NRF1 (FIG. 7C), or TRβ (FIG. 7D), along with reporter plasmids alone orin the presence of PGC-1β. For GR and TRβ transfection, ligands (Dex, 1μM dexamethasone, and 50 nM T3, respectively) were added 24 hours beforethe cells were lysed and assayed for luciferase activity.

FIG. 8 depicts the mapping of the transcriptional activation domain ofmurine PGC-1β. Full-length PGC-1β or fragments thereof were fused toGAL4-DBD and assayed for the activation of transcription from a5×UAS-luciferase reporter construct (5×UAS, five copies of the upstreamactivation sequence) in transiently transfected BOSC cells.GAL4-DBD-PGC1α fusion plasmids were included for comparison. Luciferaseactivity was expressed as fold activation over vector alone. Error barsindicate SEM of three independent experiments performed in duplicate.

FIG. 9 depicts the interaction and activation of PGC-1 by HCF. In FIG.9A, BOSC cells were transiently transfected with GAL4-PGC-1β andFLAG-HCF expression constructs as indicated. In FIG. 9B, the N-terminal380 amino acids of HCF were fused to GAL4-DBD. The resulting constructwas transiently transfected into BOSC cells alone or in the presence ofFlag-PGC-1β. Luciferase activity was expressed as fold activation overvector alone. Error bars indicate SEM of three independent experimentsperformed in duplicate.

FIGS. 10A-10B depict that PGC-1β induces mitochondrial gene expressionbut not gluconeogenesis in hepatocytes. FAO hepatoma cells (FIG. 10A) orprimary rat hepatocytes (FIG. 10B) were infected with varying doses ofrecombinant GFP, PGC-1α or PGC-1β viruses for 48 hours. Total RNA wasisolated and analyzed by Northern hybridization to examine theexpression of various genes using gene-specific probes such as PEPCK andG6Pase for gluconeogenesis and CPT-1, MCAD and Cytochrome C for fattyacid oxidation. The results indicate that PGC-1α activates bothgluconeogenesis and fatty acid oxidation as evidenced by increasedexpression of PEPCK, G6Pase, CPT-1, MCAD and Cytochrome C, but PGC-1βonly induces the mitochondrial fatty acid oxidation genes CPT-1, MCADand Cytochrome C.

FIGS. 11A-11B depict that PGC-1β induces mitochondrial biogenesis inmurine myotubes and that enzymes involved in free radical metabolism arehighly elevated in response to PGC-1α and PGC-1β. C2C12 myotubes wereinfected with recombinant adenoviruses and total RNA (FIG. 11A) andtotal DNA (FIG. 11B) was isolated and examined for gene expression andmitochondrial DNA content, respectively.

FIG. 12 depicts that PGC-1α and PGC-1β induce mitochondrial geneexpression in neuroblastoma cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel members of the family of PGC-1 molecules, referred to herein asPGC-1β nucleic acid and protein molecules. The present invention isfurther based, at least in part, on the discovery that PGC-1β isupregulated during brown fat determination/differentiation, but is notregulated in animals upon cold exposure or in brown adipose cells upontreatment with forskolin. The present invention is further based, atleast in part, on the discovery that PGC-1β expression is upregulated inthe liver during fasting. The present invention is further based, atleast in part, on the discovery that PGC-1β induces fatty acid oxidationgene expression in the liver and skeletal muscle. The present inventionis further based, at least in part, on the discovery that PGC-1β ishighly expressed in brown adipose tissue (BAT) and heart (tissues thatcontain high levels of mitochondria and substrate oxidation) and on thediscovery that PGC-1β is a potent coactivator of NRF1. The presentinvention is still further based, at least in part, on the discoverythat host cell factor (HCF), a cellular protein that is involved inherpes simplex virus (HSV) infection and cell cycle regulation (Wilson,A. C. et al. (1993) Cell 74:115-125; Wilson, A. C. et al. (1997) Mol.Cell. Biol. 17:6139-6146), is a binding partner that upregulates thetranscriptional activity of both the originally identified PGC-1(hereinafter referred to as PGC-1α), and PGC-1β. The present inventionis further based, at least in part, on the discovery that both PGC-1αand PGC-1β induce mitochondrial gene expression in neuroblastoma cells.The present invention is yet further based, at least in part, on thediscovery that both PGC-1α, and PGC-1β induce the expression of enzymesinvolved in free radical metabolism such as superoxide dismutase(Mn-SOD) and glutathione peroxidase (GPx), suggesting an important rolein the cellular defense against free radical damage.

The PGC-1β nucleic acid and protein molecules of the present inventionare useful as modulating agents in regulating a variety of cellularprocesses, e.g., cellular determination and/or differentiation (e.g.,brown adipose determination and/or differentiation), cellularmetabolism, fatty acid oxidation, mitochondrial function and/orrespiration, cellular signaling, cellular defense, and/or cellularproliferation.

Thus, the PGC-1β molecules of the present invention provide noveldiagnostic targets and therapeutic agents to control metabolicdisorders, neurological disorders and free-radical damage-relateddisorders. As used herein, the term “metabolic disorder” includes, butis not limited to, conditions, disorders, and/or diseases caused oraffected by aberrant regulation of metabolism, e.g., aberrant regulationof metabolism caused by aberrant regulation of PGC-1β expression oractivity.

For example, in one embodiment, a metabolic disorder includes a “brownadipose cell disorder”. As used herein, a “brown adipose cell disorder”includes a disease, disorder, or condition which affects a brown adiposecell or tissue. Brown adipose cell disorders include diseases,disorders, or conditions associated with aberrant thermogenesis oraberrant brown adipose cell content or function. Brown adipose celldisorders can be characterized by a misregulation (e.g., downregulationor upregulation) of PGC-1β expression or activity. Examples of brownadipose cell disorders include disorders such as obesity, overweight,anorexia, cachexia, and diabetes (e.g., type 1 diabetes, type 2diabetes, and maturity onset diabetes of the young (MODY)). Obesity isdefined as a body mass index (BMI) of 30 kg/²m or more (NationalInstitute of Health, Clinical Guidelines on the Identification,Evaluation, and Treatment of Overweight and Obesity in Adults (1998)).However, the present invention is also intended to include a disease,disorder, or condition that is characterized by a body mass index (BMI)of 25 kg/²m or more, 26 kg/²m or more, 27 kg/²m or more, 28 kg/²m ormore, 29 kg/²m or more, 29.5 kg/2m or more, or 29.9 kg/²m or more, allof which are typically referred to as overweight (National Institute ofHealth, Clinical Guidelines on the Identification, Evaluation, andTreatment of Overweight and Obesity in Adults (1998)).

Metabolic disorders also include disorders associated with aberrantglucose homeostasis, for example, diabetes (e.g., type 1 diabetes, type2 diabetes, and maturity onset diabetes of the young (MODY)) anddisorders characterized by underproduction of glucose, e.g., hepaticenzyme abnormalities which result in hypoglycemia; and hypoglycemia,e.g., secondary hypoglycemia caused by other diseases, disorders, orconditions. Metabolic disorders may also include any other disorder orcondition that is affected by abnormalities of glucose homeostasis,e.g., weight disorders such as obesity, cachexia, anorexia, anddisorders associated with insufficient insulin activity. Disordersassociated with body weight are disorders associated with abnormal bodyweight or abnormal control of body weight. As used herein, the language“diseases associated with or characterized by insufficient insulinactivity” includes disorders or diseases in which there is an abnormalutilization of glucose due to abnormal insulin function. Abnormalinsulin function includes any abnormality or impairment in insulinproduction, e.g., expression and/or transport through cellularorganelles, such as insulin deficiency resulting from, for example, lossof β cells as in IDDM (type 1 diabetes), secretion, such as impairmentof insulin secretory responses as in NIDDM (type 2 diabetes), the formof the insulin molecule itself, e.g., primary, secondary or tertiarystructure, effects of insulin on target cells, e.g., insulin-resistancein bodily tissues, e.g., peripheral tissues, and responses of targetcells to insulin. See Braunwald, E. et al. eds. Harrison's Principles ofInternal Medicine, Eleventh Edition (McGraw-Hill Book Company, New York,1987) pp. 1778-1797; Robbins, S. L. et al. Pathologic Basis of Disease,3rd Edition (W.B. Saunders Company, Philadelphia, 1984) p. 972 forfurther descriptions of abnormal insulin activity in IDDM and NIDDM andother forms of diabetes

As used herein, the term “neurological disorder” includes, but is notlimited to, conditions, disorders, and/or diseases caused or affected byaberrant regulation of brain energy metabolism, e.g., aberrantregulation of brain energy metabolism caused by aberrant regulation ofPGC-1 expression or activity. For example, in one embodiment, a“neurological disorder” includes disorders of the nervous system,including, but not limited to those involving the brain, the central andperipheral nervous system, and the interfaces between muscles and thenerves. Some examples of neurological related disorders include, withoutlimitation, Alzheimer's disease, dementias related to Alzheimer'sdisease (such as Pick's disease), Parkinson's and other Lewy diffusebody diseases, multiple sclerosis, amyotrophic lateral sclerosis,progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldtdisease. “Neurological disorders” also includes neurological disordersassociated with inflammation, e.g. stroke, traumatic injury to thebrain, traumatic injury to the spinal cord, spinal crush, central andperipheral nervous system trauma (CNS disorders).

Examples of CNS disorders such as cognitive and neurodegenerativedisorders, include, but are not limited to, Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, senile dementia,Huntington's disease, Gilles de la Tourette's syndrome, multiplesclerosis, amyotrophic lateral sclerosis, progressive supranuclearpalsy, epilepsy, and Jakob-Creutzfieldt disease; autonomic functiondisorders such as hypertension and sleep disorders, and neuropsychiatricdisorders, such as depression, schizophrenia, schizoaffective disorder,korsakoff's psychosis, mania, anxiety disorders, or phobic disorders;learning or memory disorders, e.g., amnesia or age-related memory loss,attention deficit disorder, dysthymic disorder, major depressivedisorder, mania, obsessive-compulsive disorder, psychoactive substanceuse disorders, anxiety, phobias, panic disorder, as well as bipolaraffective disorder, e.g., severe bipolar affective (mood) disorder(BP-1), and bipolar affective neurological disorders, e.g., migraine andobesity. Further CNS-related disorders include, for example, thoselisted in the American Psychiatric Association's Diagnostic andStatistical manual of Mental Disorders (DSM), the most current versionof which is incorporated herein by reference in its entirety.“Neurological disorders” also includes disorders related to free radicaldamage.

As used herein, the term “free-radical damage-related disorder”includes, but is not limited to, conditions, disorders, and/or diseasescaused or affected by aberrant regulation of free radical metabolism,e.g., aberrant regulation of free-radical metabolism caused by aberrantregulation of PGC-1 expression or activity. For example, in oneembodiment, a “free-radical damage-related disorder” includes,neurodegenerative diseases and neurodegenerative disorders such asHuntington's (HD), Parkinson's (PD), and Alzheimer's diseases, as wellas amyotrophic lateral sclerosis (ALS), commonly known as Lou Gehrig'sdisease. A “free-radical damage-related disorder” also includesbiological senescence or aging defined as an increase in the risk ofdeath which results from deleterious cellular changes produced byfree-radical reactions. These cell-damaging processes are largelyinitiated in the course of mitochondrial respiration, while life span isdetermined by the rate of damage to the mitochondria. A “free-radicaldamage-related disorder” further includes all forms of cancer and heartdisease as well as all known disorders associated with impairedfree-radical metabolism.

Because the PGC-1β molecules of the invention interact with host cellfactor (HCF), they may also provide novel diagnostic targets andtherapeutic agents to control viral disorders and/or cellularproliferation, growth, and/or differentiation disorders. Viral disordersincluded disorders, diseases, and/or conditions caused or affected byinfection of a cell or a subject by a virus, e.g., herpes simplex virus(HSV). Cellular proliferation, growth, differentiation, disordersinclude those disorders that affect cell proliferation, growth, and/ordifferentiation processes. As used herein, a “cellular proliferation,growth, and/or differentiation process” is a process by which a cellincreases in number, size or content, by which a cell develops aspecialized set of characteristics which differ from that of othercells, or by which a cell moves closer to or further from a particularlocation or stimulus. Thus, the PGC-1β molecules may modulate cellulargrowth, and/or differentiation and may play a role in disorderscharacterized by aberrantly regulated growth, and/or differentiation.Such disorders include cancer, e.g., carcinomas, sarcomas, leukemias,and lymphomas, and in particular, cancers caused by infection with avirus, e.g., herpes simplex virus (HSV); tumor angiogenesis andmetastasis; skeletal dysplasia; hepatic disorders; and hematopoieticand/or myeloproliferative disorders.

PGC-1β-associated or related disorders also include disorders affectingtissues in which PGC-1β protein is expressed, e.g., brown adiposetissue, white adipose tissue, liver, and/or heart.

As used herein, “brown adipose cell activity” includes an activityexerted by a brown adipose cell, or an activity that takes place in abrown adipose cell. For example, such activities include cellularprocesses that contribute to the physiological role of brown adiposecells, such as brown adipose cell differentiation and mitochondrialactivity and include, but are not limited to, cell proliferation,differentiation, growth, migration, programmed cell death, uncoupledmitochondrial respiration, and thermogenesis.

The term “family” when referring to the protein and nucleic acidmolecules of the invention is intended to mean two or more proteins ornucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence homology as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin, as well as other,distinct proteins of human origin or alternatively, can containhomologues of non-human origin, e.g., monkey proteins. Members of afamily may also have common functional characteristics.

For example, a member of the family of PGC-1β molecules of the inventioncomprises at least one “LXXLL motif” in the protein or correspondingnucleic acid molecule. As used herein, an “LXXLL motif” refers to amotif wherein X can be any amino acid and which mediates an interactionbetween an nuclear receptor and a coactivator (Heery et al. (1997)Nature 397:733-736; Torchia et al. (1997) Nature 387:677-684). In apreferred embodiment, a PGC-1β protein has at least two or three LXXLLmotifs. Three LXXLL motifs were identified in the amino acid sequence ofmouse PGC-1β at about residues 140-144, 156-160, and 343-347 of SEQ IDNO:2 (FIG. 2). Two LXXLL motifs were identified in the amino acidsequence of human PGC-1β at about residues 144-148 and 331-335 of SEQ IDNO:5 (FIG. 4).

In another embodiment of the invention, a PGC-1β molecule of theinvention comprises at least one “RNA recognition motif” or “RRM” in theprotein or corresponding nucleic acid molecule. As used interchangeablyherein, an “RNA recognition motif” or “RRM” is an amino acid sequencewhich can bind an RNA molecule or a single stranded DNA molecule. In apreferred embodiment an RRM is found near the C-terminus of a PGC-1βprotein and comprises about 50-160, 60-150, 70-140, 80-130, 90-120, orpreferably about 108 amino acid residues. RRMs are described in Lodish,H., Darnell, J., and Baltimore, D. Molecular Cell Biology, 3rd ed. (W.H.Freeman and Company, New York, N.Y., 1995). An RRM was identified in theamino acid sequence of mouse PGC-1β at about residues 894-958 of SEQ IDNO:2 (FIG. 2). An RRM was identified in the amino acid sequence of humanPGC-1β at about residues 889-953 of SEQ ID NO:5 (FIG. 5).

In another embodiment, a PGC-1β molecule of the invention comprises atleast one “activation domain” or “AD” in the protein or correspondingnucleic acid molecule. As used interchangeably herein, an “activationdomain” or “AD” is a protein domain which has autonomous transcriptionalactivity when fused to a heterologous DNA binding domain. In a preferredembodiment, an AD is located N-terminal 220 amino acid residues of aPGC-1β protein and comprises about 50-375, 75-350, 100-325, 125-300,150-275, 175-250, 200-225, or about 220 amino acid residues. An AD wasidentified in the amino acid sequence of mouse PGC-1β at about aminoacid residues 1-220 of SEQ ID NO:2.

In still another embodiment, a PGC-1β molecule of the inventioncomprises at least one “host cell factor binding motif” in the proteinor corresponding nucleic acid molecule. As used interchangeably herein,a “host cell factor binding motif”, “host cell factor binding site”, or“HBM” includes an amino acid motif capable of mediating the interactionof a PGC-1 molecule and host cell factor (HCF), a protein involved inthe regulation of cell cycle progression and the assembly of amultiprotein transcriptional complex during herpes simplex virus (HSV)infection (Freiman, R. N. and Herr, W. (1997) Genes Dev. 11:3122-3127;Andersson, U. and Scarpulla, R. C. (2001) Mol. Cell. Biol.21:3738-3749). In a preferred embodiment, an HBM has the consensussequence [D/E]-H-X-Y, wherein [D/E] indicates either D or E at theindicated position, and wherein X indicates any amino acid at theindicated position. An HBM was identified in the amino acid sequence ofmouse PGC-1β at about residues 683-686 of SEQ ID NO:2 (FIG. 2). An HBMwas also identified in the amino acid sequence of human PGC-1β at aboutresidues 677-680 of SEQ ID NO:5 (FIG. 5). An HBM was also identified inthe amino acid sequence of mouse PGC-1α at about residues 382-385 of SEQID NO:9, and in the amino acid sequence of human PGC-1α at aboutresidues 383-386 of SEQ ID NO:11.

In another embodiment, a PGC-1β molecule of the invention comprises atleast one “glutamic/aspartic acid rich acidic domain” in the protein orcorresponding nucleic acid molecule. As used herein, a“glutamic/aspartic acid rich acidic domain” includes a protein domain ofabout 10-40, 12-35, 14-30, 16-25, or preferably about 18, 21, or 23amino acid residues. Glutamic/aspartic acid rich acidic domains arefound in proteins that regulate diverse biological processes, includingtranscription, assembly of RNA-protein complexes, and modification ofprotein structure. In a preferred embodiment, all of the amino acidresidues in a glutamic/aspartic acid rich acidic domain are acidicresidues (e.g., glutamic acid or aspartic acid). In other embodiments aglutamic/aspartic acid rich acidic domain may have at least 1, 2, 3, 4,5, 6, 7, or 8 amino acid residues which are not acidic. Preferably, aPGC-1β molecule comprises at least two glutamic/aspartic acid richacidic domains. Two glutamic/aspartic acid rich acidic domains wereidentified in the amino acid sequence of mouse PGC-1β at about residues429-451 and 798-815 of SEQ ID NO:2. Two glutamic/aspartic acid richacidic domains were identified in the amino acid sequence of humanPGC-1β at about residues 418-438 and 793-810 of SEQ ID NO:5.

In a preferred embodiment, the PGC-1β molecules of the invention includeat least one or more of the following domains: an LXXLL motif, an RRM,an AD, an HBM, and/or a glutamic/aspartic acid rich acidic domain.

Isolated proteins of the present invention, preferably PGC-1β proteins,have an amino acid sequence sufficiently identical to the amino acidsequence of SEQ ID NO:2 or 5, or are encoded by a nucleotide sequencesufficiently identical to SEQ ID NO:1, 3, 4, or 6. As used herein, theterm “sufficiently identical” refers to a first amino acid or nucleotidesequence which contains a sufficient or minimum number of identical orequivalent (e.g., an amino acid residue which has a similar side chain)amino acid residues or nucleotides to a second amino acid or nucleotidesequence such that the first and second amino acid or nucleotidesequences share common structural domains or motifs and/or a commonfunctional activity. For example, amino acid or nucleotide sequenceswhich share common structural domains have at least 30%, 40%, or 50%homology, preferably 60% homology, more preferably 70%-80%, and evenmore preferably 90-95% homology across the amino acid sequences of thedomains and contain at least one and preferably two structural domainsor motifs, are defined herein as sufficiently identical. Furthermore,amino acid or nucleotide sequences which share at least 30%, 40%, or50%, preferably 60%, more preferably 70-80%, or 90-95% homology andshare a common functional activity are defined herein as sufficientlyidentical.

As used interchangeably herein, an “PGC-1β activity”, “biologicalactivity of PGC-1β” or “functional activity of PGC-1β”, refers to anactivity exerted by a PGC-1β protein, polypeptide or nucleic acidmolecule on a PGC-1β responsive cell or tissue, or on a PGC-1β proteinsubstrate, as determined in vivo, or in vitro, according to standardtechniques. In one embodiment, a PGC-1β activity is a direct activity,such as an association with a PGC-1β-target molecule. As used herein, a“target molecule” or “binding partner” is a molecule with which a PGC-1βprotein binds or interacts in nature, such that PGC-1β-mediated functionis achieved. In an exemplary embodiment, a PGC-1β target molecule is anuclear receptor (e.g., HNF4α, PPARα, retinoic acid receptor α (RARα),thyroid hormone receptor β (TRβ), and glucocorticoid receptor (GR)),host cell factor (HCF), nuclear respiratory factor 1 (NRF1), or a basaltranscription factor. Alternatively, a PGC-1β activity is an indirectactivity, such as a cellular signaling activity mediated by interactionof the PGC-1β protein with a PGC-1β target molecule. The biologicalactivities of PGC-1β are described herein. For example, the PGC-1βproteins of the present invention can have one or more of the followingactivities: 1) interaction with a nuclear receptor (e.g., HNF4α, PPARα,retinoic acid receptor α (RARα), thyroid hormone receptor β (TRβ), orglucocorticoid receptor (GR)); 2) interaction with HCF; 3) interactionwith NRF1; 4) interaction with a basal transcription factor; 5)modulation of the activity, e.g., the transcriptional activity, of anuclear receptor and/or NRF1; 6) modulation of brown adipose celldetermination and/or differentiation; 7) modulation on intra- orinter-cellular signaling; 8) modulation of viral infection (e.g., viainteraction with HCF); 9) modulation of cellular proliferation; 10)modulation of metabolism; 11) modulation of mitochondrial activityand/or biogenesis; and 12) modulation of fatty acid β-oxidation.

Accordingly, another embodiment of the invention features isolatedPGC-1β proteins and polypeptides having a PGC-1β activity. Otherpreferred proteins are PGC-1β proteins having one or more of thefollowing domains: an LXXLL motif, an RRM, an AD, an HBM, and/or aglutamic/aspartic acid rich acidic domain, preferably, a PGC-1βactivity.

Additional preferred proteins have at least one or more of an LXXLLmotif, an RRM, an AD, an HBM, and/or a glutamic/aspartic acid richacidic domain, and are, preferably, encoded by a nucleic acid moleculehaving a nucleotide sequence which hybridizes under stringenthybridization conditions to a complement of a nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, 3, 4, or 6.

The nucleotide sequence of the isolated mouse PGC-1β cDNA is shown inFIGS. 1A-1B and in SEQ ID NO:1, and the predicted amino acid sequence ofthe mouse PGC-1β polypeptide is shown in FIG. 2 and in SEQ ID NO:2. Thenucleotide sequence of the isolated human PGC-1β cDNA is shown in FIGS.3A-3B and in SEQ ID NO:4, and the predicted amino acid sequence of thehuman PGC-1β polypeptide is shown in FIG. 4 and in SEQ ID NO:5. Thesedeposits will be maintained under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thatdeposits are required under 35 U.S.C. §112.

The mouse PGC-1β gene, which is approximately 3664 nucleotides inlength, encodes a protein having a molecular weight of approximately111.5 kD and is approximately 1014 amino acid residues in length. Thehuman PGC-1β gene, which is approximately 3030 nucleotides in length,encodes a protein having a molecular weight of approximately 111.0 kDand is approximately 1009 amino acid residues in length.

Various aspects of the invention are described in further detail in thefollowing subsections:

I. ISOLATED NUCLEIC ACID MOLECULES

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode PGC-1β proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify PGC-1β-encoding nucleic acid molecules (e.g., PGC-1βmRNA) and fragments for use as PCR primers for the amplification ormutation of PGC-1β nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecules ofthe present invention can be single-stranded or double-stranded, butpreferably are double-stranded DNA.

The term “isolated nucleic acid molecule” includes nucleic acidmolecules which are separated from other nucleic acid molecules whichare present in the natural source of the nucleic acid. For example, withregards to genomic DNA, the term “isolated” includes nucleic acidmolecules which are separated from the chromosome with which the genomicDNA is naturally associated. Preferably, an “isolated” nucleic acid isfree of sequences which naturally flank the nucleic acid (i.e.,sequences located at the 5′ and 3′ ends of the nucleic acid) in thegenomic DNA of the organism from which the nucleic acid is derived. Forexample, in various embodiments, the isolated PGC-1β nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of nucleotide sequences which naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. Moreover, an “isolated” nucleic acid molecule, such as a cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, 3, 4, or 6, or aportion thereof, can be isolated using standard molecular biologytechniques and the sequence information provided herein. Using all orportion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, or 6, as ahybridization probe, PGC-1β nucleic acid molecules can be isolated usingstandard hybridization and cloning techniques (e.g., as described inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO:1, 3, 4, or 6, can be isolated by the polymerase chain reaction(PCR) using synthetic oligonucleotide primers designed based upon thesequence of SEQ ID NO:1, 3, 4, or 6.

A nucleic acid of the invention can be amplified using cDNA, mRNA or,alternatively, genomic DNA as a template and appropriate oligonucleotideprimers according to standard PCR amplification techniques. The nucleicacid so amplified can be cloned into an appropriate vector andcharacterized by DNA sequence analysis. Furthermore, oligonucleotidescorresponding to PGC-1β nucleotide sequences can be prepared by standardsynthetic techniques, e.g., using an automated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, 3, 4,or 6. This cDNA may comprise sequences encoding the mouse PGC-1β protein(i.e., “the coding region”, from nucleotides 65-3106), as well as 5′untranslated sequences (nucleotides 1-64) and 3′ untranslated sequences(nucleotides 3107-3664) of SEQ ID NO:1. Alternatively, the nucleic acidmolecule can comprise only the coding region of SEQ ID NO:1 (e.g.,nucleotides 65-3106, corresponding to SEQ ID NO:3). Accordingly, inanother embodiment, an isolated nucleic acid molecule of the inventioncomprises SEQ ID NO:3 and nucleotides 1-65 of SEQ ID NO:1. In yetanother embodiment, the isolated nucleic acid molecule comprises SEQ IDNO:3 and nucleotides 3107-3664 of SEQ ID NO:1. In yet anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:1 or SEQ ID NO:3. In still anotherembodiment, the nucleic acid molecule can comprise the coding region ofSEQ ID NO:1 (e.g., nucleotides 65-3106, corresponding to SEQ ID NO:3),as well as a stop codon (e.g., nucleotides 3107-3109 of SEQ ID NO:1).

This cDNA may comprise sequences encoding the human PGC-1β protein(i.e., “the coding region”, from nucleotides 1-3027), as well as a stopcodon (nucleotides 3028-3030) of SEQ ID NO:4. Alternatively, the nucleicacid molecule can comprise only the coding region of SEQ ID NO:4 (e.g.,nucleotides 1-3027, corresponding to SEQ ID NO:6). In yet anotherembodiment, the nucleic acid molecule consists of the nucleotidesequence set forth as SEQ ID NO:4 or SEQ ID NO:6.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, or a portionof any of these nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, 3, 4, or6, is one which is sufficiently complementary to the nucleotide sequenceshown in SEQ ID NO:1, 3, 4, or 6, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, respectively,thereby forming a stable duplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,99.99% or more identical to the entire length of the nucleotide sequenceshown in SEQ ID NO:1, 3, 4, or 6, or a portion of any of thesenucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, 3, 4, or 6, forexample, a fragment which can be used as a probe or primer or a fragmentencoding a portion of a PGC-1β protein, e.g., a biologically activeportion of a PGC-1β protein. The nucleotide sequences determined fromthe cloning of the PGC-1β genes allow for the generation of probes andprimers designed for use in identifying and/or cloning other PGC-1βfamily members, as well as PGC-1β homologues from other species. Theprobe/primer typically comprises substantially purified oligonucleotide.The oligonucleotide typically comprises a region of nucleotide sequencethat hybridizes under stringent conditions to at least about 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense sequence of SEQ IDNO:1, 3, 4, or 6, of an anti-sense sequence of SEQ ID NO:1, 3, 4, or 6,or of a naturally occurring allelic variant or mutant of SEQ ID NO:1, 3,4, or 6. In one embodiment, a nucleic acid molecule of the presentinvention comprises a nucleotide sequence which is greater than 50, 75,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 457, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850,1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450,2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050,3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600 or morenucleotides in length and hybridizes under stringent hybridizationconditions to a nucleic acid molecule of SEQ ID NO:1, 3, 4, or 6.

Probes based on the PGC-1β nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a PGC-1β protein, such as by measuring a levelof a PGC-1β-encoding nucleic acid in a sample of cells from a subjecte.g., detecting PGC-1β mRNA levels or determining whether a genomicPGC-1β gene has been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aPGC-1β protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, 3, 4, or 6, which encodes a polypeptide havinga PGC-1β biological activity (the biological activities of the PGC-1βproteins are described herein), expressing the encoded portion of thePGC-1β protein (e.g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of the PGC-1β protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, 3, 4, or 6, due todegeneracy of the genetic code and thus encode the same PGC-1β proteinsas those encoded by the nucleotide sequence shown in SEQ ID NO:1, 3, 4,or 6, an isolated nucleic acid molecule of the invention has anucleotide sequence encoding a protein having an amino acid sequenceshown in SEQ ID NO:2 or 5.

In addition to the PGC-1β nucleotide sequences shown in SEQ ID NO:1, 3,4, or 6, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the PGC-1β proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the PGC-1β genes may existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a PGC-1βprotein, preferably a mammalian PGC-1β protein, and can further includenon-coding regulatory sequences, and introns.

Allelic variants of human PGC-1β include both functional andnon-functional PGC-1β proteins. Functional allelic variants arenaturally occurring amino acid sequence variants of the human PGC-1βprotein that maintain the ability to bind a PGC-1β target molecule andor modulate transcriptional and/or cell differentiation and/orproliferation mechanisms. Functional allelic variants will typicallycontain only conservative substitution of one or more amino acids of SEQID NO:2 or 5, or substitution, deletion or insertion of non-criticalresidues in non-critical regions of the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the mouse or human PGC-1β proteins that do not havethe ability to either bind a PGC-1β target molecule and/or modulate anyof the PGC-1β activities described herein. Non-functional allelicvariants will typically contain a non-conservative substitution, adeletion, or insertion or premature truncation of the amino acidsequence of SEQ ID NO:2 or 5, or a substitution, insertion or deletionin critical residues or critical regions of the protein.

The present invention further provides non-human orthologues of themouse or human PGC-1β proteins. Orthologues of the mouse or human PGC-1βproteins are proteins that are isolated from non-human organisms andpossess the same PGC-1β activities of the mouse or human PGC-1βproteins, as described herein. Orthologues of the mouse or human PGC-1βproteins can readily be identified as comprising an amino acid sequencethat is substantially identical to SEQ ID NO:2 or 5.

Moreover, nucleic acid molecules encoding other PGC-1β family membersand, thus, which have a nucleotide sequence which differs from thePGC-1β sequences of SEQ ID NO:1, 3, 4, or 6, are intended to be withinthe scope of the invention. For example, another PGC-1β cDNA can beidentified based on the nucleotide sequence of mouse or human PGC-1β.Moreover, nucleic acid molecules encoding PGC-1β proteins from differentspecies, and which, thus, have a nucleotide sequence which differs fromthe PGC-1β sequences of SEQ ID NO:1, 3, 4, or 6, are intended to bewithin the scope of the invention. For example, a mouse PGC-1β cDNA canbe identified based on the nucleotide sequence of a mouse or humanPGC-1β.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the PGC-1β cDNAs of the invention can be isolated based ontheir homology to the PGC-1β nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions. Nucleic acid molecules corresponding tonatural allelic variants and homologues of the PGC-1β cDNAs of theinvention can further be isolated by mapping to the same chromosome orlocus as the PGC-1β gene.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30, 35, 40, 45, 50 or morenucleotides in length and hybridizes under stringent conditions to thenucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,3, 4, or 6. In other embodiment, the nucleic acid is at least 50, 75,100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 457, 500, 550, 600,650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250,1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850,1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400, 2450,2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050,3100, 3150, 3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600 or morenucleotides in length.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences that are significantly identical orhomologous to each other remain hybridized to each other. Preferably,the conditions are such that sequences at least about 70%, morepreferably at least about 80%, even more preferably at least about 85%or 90% identical to each other remain hybridized to each other. Suchstringent conditions are known to those skilled in the art and can befound in Current Protocols in Molecular Biology, Ausubel et al., eds.,John Wiley & Sons, Inc. (1995), sections 2, 4, and 6. Additionalstringent conditions can be found in Molecular Cloning: A LaboratoryManual, Sambrook et al., Cold Spring Harbor Press, Cold Spring Harbor,N.Y. (1989), chapters 7, 9, and 11. A preferred, non-limiting example ofstringent hybridization conditions includes hybridization in 4× sodiumchloride/sodium citrate (SSC), at about 65-70° C. (or alternativelyhybridization in 4×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 1×SSC, at about 65-70° C. A preferred,non-limiting example of highly stringent hybridization conditionsincludes hybridization in 1×SSC, at about 65-70° C. (or alternativelyhybridization in 1×SSC plus 50% formamide at about 42-50° C.) followedby one or more washes in 0.3×SSC, at about 65-70° C. A preferred,non-limiting example of reduced stringency hybridization conditionsincludes hybridization in 4×SSC, at about 50-60° C. (or alternativelyhybridization in 6×SSC plus 50% formamide at about 40-45° C.) followedby one or more washes in 2×SSC, at about 50-60° C. Ranges intermediateto the above-recited values, e.g., at 65-70° C. or at 42-50° C. are alsointended to be encompassed by the present invention. SSPE (1×SSPE is0.15M NaCl, 10 mM NaH₂PO₄, and 1.25 mM EDTA, pH 7.4) can be substitutedfor SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in thehybridization and wash buffers; washes are performed for 15 minutes eachafter hybridization is complete. The hybridization temperature forhybrids anticipated to be less than 50 base pairs in length should be5-10° C. less than the melting temperature (T_(m)) of the hybrid, whereT_(m) is determined according to the following equations. For hybridsless than 18 base pairs in length, T_(m)(° C.)=2(# of A+T bases)+4(# ofG+C bases). For hybrids between 18 and 49 base pairs in length, T_(m)(°C.)=81.5+16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N), where N is the number ofbases in the hybrid, and [Na⁺] is the concentration of sodium ions inthe hybridization buffer ([Na⁺] for 1×SSC=0.165 M). It will also berecognized by the skilled practitioner that additional reagents may beadded to hybridization and/or wash buffers to decrease non-specifichybridization of nucleic acid molecules to membranes, for example,nitrocellulose or nylon membranes, including but not limited to blockingagents (e.g., BSA or salmon or herring sperm carrier DNA), detergents(e.g., SDS), chelating agents (e.g., EDTA), Ficoll, PVP and the like.When using nylon membranes, in particular, an additional preferred,non-limiting example of stringent hybridization conditions ishybridization in 0.25-0.5M NaH₂PO₄, 7% SDS at about 65° C., followed byone or more washes at 0.02M NaH₂PO₄, 1% SDS at 65° C. (see e.g., Churchand Gilbert (1984) Proc. Natl. Acad. Sci. USA 81:1991-1995), oralternatively 0.2×SSC, 1% SDS.

Preferably, an isolated nucleic acid molecule of the invention thathybridizes under stringent conditions to the sequence of SEQ ID NO:1, 3,4, or 6, and corresponds to a naturally-occurring nucleic acid molecule.As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the PGC-1βsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, 3, 4, or 6, thereby leading tochanges in the amino acid sequence of the encoded PGC-1β proteins,without altering the functional ability of the PGC-1β proteins. Forexample, nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, 3, 4, or 6. A “non-essential” amino acid residue is a residuethat can be altered from the wild-type sequence of PGC-1β (e.g., thesequence of SEQ ID NO:2 or 5) without altering the biological activity,whereas an “essential” amino acid residue is required for biologicalactivity. For example, amino acid residues that are conserved among thePGC-1β proteins of the present invention, e.g., those present in anactivation domain, an LXXLL motif, or an HBM, are predicted to beparticularly unamenable to alteration. Furthermore, additional aminoacid residues that are conserved between the PGC-1β proteins of thepresent invention and other members of the PGC-1 family are not likelyto be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PGC-1β proteins that contain changes in amino acidresidues that are not essential for activity. Such PGC-1β proteinsdiffer in amino acid sequence from SEQ ID NO:2 or 5, yet retainbiological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%,99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99% or moreidentical to SEQ ID NO:2 or 5.

An isolated nucleic acid molecule encoding a PGC-1β protein identical tothe protein of SEQ ID NO:2 or 5 can be created by introducing one ormore nucleotide substitutions, additions or deletions into thenucleotide sequence of SEQ ID NO:1, 3, 4, or 6, such that one or moreamino acid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced into SEQ ID NO:1, 3, 4, or6, by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis. Preferably, conservative amino acidsubstitutions are made at one or more predicted non-essential amino acidresidues. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a predicted nonessential amino acid residue in aPGC-1β protein is preferably replaced with another amino acid residuefrom the same side chain family. Alternatively, in another embodiment,mutations can be introduced randomly along all or part of a PGC-1βcoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for PGC-1β biological activity to identifymutants that retain activity. Following mutagenesis of SEQ ID NO:1, 3,4, or 6, the encoded protein can be expressed recombinantly and theactivity of the protein can be determined.

In a preferred embodiment, a mutant PGC-1β protein can be assayed forthe ability to interact with and/or coactivate a nuclear receptor, HCF,and/or NRF1, for the ability to modulate brown adipose celldifferentiation, and/or for the ability to modulate mitochondrialactivity and/or biogenesis.

In addition to the nucleic acid molecules encoding PGC-1β proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PGC-1β coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding a PGC-1β.The term “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues (e.g.,the coding region of mouse PGC-1β corresponds to SEQ ID NO:3, and thecoding region of human PGC-1β corresponds to SEQ ID NO:6). In anotherembodiment, the antisense nucleic acid molecule is antisense to a“noncoding region” of the coding strand of a nucleotide sequenceencoding PGC-1β. The term “noncoding region” refers to 5′ and 3′sequences which flank the coding region that are not translated intoamino acids (i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding PGC-1β disclosed herein(e.g., SEQ ID NO:3 or 6), antisense nucleic acids of the invention canbe designed according to the rules of Watson and Crick base pairing. Theantisense nucleic acid molecule can be complementary to the entirecoding region of PGC-1β mRNA, but more preferably is an oligonucleotidewhich is antisense to only a portion of the coding or noncoding regionof PGC-1β mRNA. For example, the antisense oligonucleotide can becomplementary to the region surrounding the translation start site ofPGC-1β mRNA. An antisense oligonucleotide can be, for example, about 5,10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length. An antisensenucleic acid of the invention can be constructed using chemicalsynthesis and enzymatic ligation reactions using procedures known in theart. For example, an antisense nucleic acid (e.g., an antisenseoligonucleotide) can be chemically synthesized using naturally occurringnucleotides or variously modified nucleotides designed to increase thebiological stability of the molecules or to increase the physicalstability of the duplex formed between the antisense and sense nucleicacids, e.g., phosphorothioate derivatives and acridine substitutednucleotides can be used. Examples of modified nucleotides which can beused to generate the antisense nucleic acid include 5-fluorouracil,5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine,4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PGC-1βprotein to thereby inhibit expression of the protein, e.g., byinhibiting transcription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids Res. 15:6625-6641). Theantisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haseloff and Gerlach(1988) Nature 334:585-591)) can be used to catalytically cleave PGC-1βmRNA transcripts to thereby inhibit translation of PGC-1β mRNA. Aribozyme having specificity for a PGC-1β-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a PGC-1β cDNA disclosedherein (i.e., SEQ ID NO:1, 3, 4, or 6). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a PGC-1β-encoding mRNA. See, e.g., Cech et al. U.S.Pat. No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742.Alternatively, PGC-1β mRNA can be used to select a catalytic RNA havinga specific ribonuclease activity from a pool of RNA molecules. See,e.g., Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, PGC-1β gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of thePGC-1β (e.g., the PGC-1β promoter and/or enhancers) to form triplehelical structures that prevent transcription of the PGC-1β gene intarget cells. See generally, Helene, C. (1991) Anticancer Drug Des.6(6):569-84; Helene, C. et al. (1992) Ann. N.Y. Acad. Sci. 660:27-36;and Maher, L. J. (1992) Bioessays 14(12):807-15.

In yet another embodiment, the PGC-1β nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup, B. and Nielsen, P. E. (1996) Bioorg.Med. Chem. 4(l):5-23). As used herein, the terms “peptide nucleic acids”or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which thedeoxyribose phosphate backbone is replaced by a pseudopeptide backboneand only the four natural nucleobases are retained. The neutral backboneof PNAs has been shown to allow for specific hybridization to DNA andRNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup and Nielsen (1996) supra andPerry-O'Keefe et al. (1996) Proc. Natl. Acad. Sci. USA 93:14670-675.

PNAs of PGC-1β nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of PGC-1β nucleic acid molecules can also be used inthe analysis of single base pair mutations in a gene, (e.g., byPNA-directed PCR clamping); as ‘artificial restriction enzymes’ whenused in combination with other enzymes, (e.g., Hyrup and Nielsen (1996)supra)); or as probes or primers for DNA sequencing or hybridization(Hyrup and Nielsen (1996) supra; Perry-O'Keefe et al. (1996) supra).

In another embodiment, PNAs of PGC-1β can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of PGC-1β nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup and Nielsen (1996) supra). The synthesis of PNA-DNA chimeras canbe performed as described in Hyrup and Nielsen (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17):3357-63. For example, a DNAchain can be synthesized on a solid support using standardphosphoramidite coupling chemistry and modified nucleoside analogs,e.g., 5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, canbe used as a between the PNA and the 5′ end of DNA (Mag, M. et al.(1989) Nucleic Acids Res. 17:5973-88). PNA monomers are then coupled ina stepwise manner to produce a chimeric molecule with a 5′ PNA segmentand a 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett.5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO 88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO 89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Biotechniques 6:958-976) or intercalating agents (see,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

Alternatively, the expression characteristics of an endogenous PGC-1βgene within a cell line or microorganism may be modified by inserting aheterologous DNA regulatory element into the genome of a stable cellline or cloned microorganism such that the inserted regulatory elementis operatively linked with the endogenous PGC-1β gene. For example, anendogenous PGC-1β gene which is normally “transcriptionally silent”,i.e., a PGC-1β gene which is normally not expressed, or is expressedonly at very low levels in a cell line or microorganism, may beactivated by inserting a regulatory element which is capable ofpromoting the expression of a normally expressed gene product in thatcell line or microorganism. Alternatively, a transcriptionally silent,endogenous PGC-1β gene may be activated by insertion of a promiscuousregulatory element that works across cell types.

A heterologous regulatory element may be inserted into a stable cellline or cloned microorganism, such that it is operatively linked with anendogenous PGC-1β gene, using techniques, such as targeted homologousrecombination, which are well known to those of skill in the art, anddescribed, e.g., in Chappel, U.S. Pat. No. 5,272,071; PCT publicationNo. WO 91/06667, published May 16, 1991.

II. Isolated PGC-1β Proteins and Anti-PGC-1β Antibodies

One aspect of the invention pertains to isolated PGC-1β proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-PGC-1β antibodies. In oneembodiment, native PGC-1β proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, PGC-1β proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a PGC-1β protein or polypeptide can be synthesizedchemically using standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thePGC-1β protein is derived, or substantially free from chemicalprecursors or other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations ofPGC-1β protein in which the protein is separated from cellularcomponents of the cells from which it is isolated or recombinantlyproduced. In one embodiment, the language “substantially free ofcellular material” includes preparations of PGC-1β protein having lessthan about 30% (by dry weight) of non-PGC-1β protein (also referred toherein as a “contaminating protein”), more preferably less than about20% of non-PGC-1β protein, still more preferably less than about 10% ofnon-PGC-1β protein, and most preferably less than about 5% non-PGC-1βprotein. When the PGC-1β protein or biologically active portion thereofis recombinantly produced, it is also preferably substantially free ofculture medium, i.e., culture medium represents less than about 20%,more preferably less than about 10%, and most preferably less than about5% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of PGC-1β protein in which the proteinis separated from chemical precursors or other chemicals which areinvolved in the synthesis of the protein. In one embodiment, thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of PGC-1β protein having less than about 30% (bydry weight) of chemical precursors or non-PGC-1β chemicals, morepreferably less than about 20% chemical precursors or non-PGC-1βchemicals, still more preferably less than about 10% chemical precursorsor non-PGC-1β chemicals, and most preferably less than about 5% chemicalprecursors or non-PGC-1β chemicals.

As used herein, a “biologically active portion” of a PGC-1β proteinincludes a fragment of a PGC-1β protein which participates in aninteraction between a PGC-1β molecule and a non-PGC-1β molecule.Biologically active portions of a PGC-1β protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the PGC-1β protein, e.g., the amino acidsequence shown in SEQ ID NO:2 or 5, which include less amino acids thanthe full length PGC-1β proteins, and exhibit at least one activity of aPGC-1β protein. Typically, biologically active portions comprise adomain or motif with at least one activity of the PGC-1β protein, e.g.,interaction with and/or coactivation of a nuclear receptor, and/or brownadipose cell differentiation. A biologically active portion of a PGC-1βprotein can be a polypeptide which is, for example, 25, 50, 75, 100,125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000 or more amino acids in length.Biologically active portions of a PGC-1β protein can be used as targetsfor developing agents which modulate a PGC-1β mediated activity, e.g., atranscriptional response.

It is to be understood that a preferred biologically active portion of aPGC-1β protein of the present invention may contain one or more of thefollowing domains: an LXXLL motif, an RRM, an AD, an HBM, and/or aglutamic/aspartic acid rich acidic domain. Moreover, other biologicallyactive portions, in which other regions of the protein are deleted, canbe prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native PGC-1β protein.

In a preferred embodiment, the PGC-1β protein has an amino acid sequenceshown in SEQ ID NO:2 or 5. In other embodiments, the PGC-1β protein issubstantially identical to SEQ ID NO:2 or 5, and retains the functionalactivity of the protein of SEQ ID NO:2 or 5, yet differs in amino acidsequence due to natural allelic variation or mutagenesis, as describedin detail in subsection I above. Accordingly, in another embodiment, thePGC-1β protein is a protein which comprises an amino acid sequence atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%,99.99% or more identical to SEQ ID NO:2 or 5.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-identical sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the mouse PGC-1βamino acid sequence of SEQ ID NO:2 having 1014 amino acid residues, atleast 304, preferably at least 406, more preferably at least 507, evenmore preferably at least 608, and even more preferably at least 710,811, 913 or more amino acid residues are aligned; when aligning a secondsequence to the PGC-1β amino acid sequence of SEQ ID NO:5 having 1009amino acid residues, at least 303, preferably at least 404, morepreferably at least 505, even more preferably at least 605, and evenmore preferably at least 706, 807, 908 or more amino acid residues arealigned). The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available onlinethrough the website of the Genetics Computer Group), using either aBlosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10,8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage (available online through the website of the Genetics ComputerGroup), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70,or 80 and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment,the percent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of Meyers, E. and Miller, W. (Comput.Appl. Biosci. 4:11-17 (1988)) which has been incorporated into the ALIGNprogram (version 2.0 or 2.0U), using a PAM120 weight residue table, agap length penalty of 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to PGC-1β nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=100,wordlength=3 to obtain amino acid sequences homologous to PGC-1β proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See the website of the NationalCenter for Biotechnology Information.

The invention also provides PGC-1β chimeric or fusion proteins. As usedherein, a PGC-1β “chimeric protein” or “fusion protein” comprises aPGC-1β polypeptide operatively linked to a non-PGC-1β polypeptide. An“PGC-1β polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a PGC-1β molecule, whereas a “non-PGC-1βpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to a protein which is not substantially homologous to thePGC-1β protein, e.g., a protein which is different from the PGC-1βprotein and which is derived from the same or a different organism.Within a PGC-1β fusion protein the PGC-1β polypeptide can correspond toall or a portion of a PGC-1β protein. In a preferred embodiment, aPGC-1β fusion protein comprises at least one biologically active portionof a PGC-1β protein. In another preferred embodiment, a PGC-1β fusionprotein comprises at least two biologically active portions of a PGC-1βprotein. Within the fusion protein, the term “operatively linked” isintended to indicate that the PGC-1β polypeptide and the non-PGC-1βpolypeptide are fused in-frame to each other. The non-PGC-1β polypeptidecan be fused to the N-terminus or C-terminus of the PGC-1β polypeptide.

For example, in one embodiment, the fusion protein is a GST-PGC-1βfusion protein in which the PGC-1β sequences are fused to the C-terminusof the GST sequences. Such fusion proteins can facilitate thepurification of recombinant PGC-1β.

In another embodiment, the fusion protein is a PGC-1β protein containinga heterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of PGC-1β canbe increased through use of a heterologous signal sequence.

The PGC-1β fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. ThePGC-1β fusion proteins can be used to affect the bioavailability of aPGC-1β target molecule. Use of PGC-1β fusion proteins may be usefultherapeutically for the treatment of disorders caused by, for example,(i) aberrant modification or mutation of a gene encoding a PGC-1βprotein; (ii) mis-regulation of the PGC-1β gene; and (iii) aberrantpost-translational modification of a PGC-1β protein.

Moreover, the PGC-1β-fusion proteins of the invention can be used asimmunogens to produce anti-PGC-1 antibodies in a subject, to purifyPGC-1 P ligands and in screening assays to identify molecules whichinhibit the interaction of PGC-1 P with a PGC-1 substrate.

Preferably, a PGC-1 P chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). APGC-1β-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the PGC-1βprotein.

The present invention also pertains to variants of the PGC-1β proteinswhich function as either PGC-1β agonists (mimetics) or as PGC-1βantagonists. Variants of the PGC-1β proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a PGC-1βprotein. An agonist of the PGC-1β proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a PGC-1β protein. An antagonist of a PGC-1β proteincan inhibit one or more of the activities of the naturally occurringform of the PGC-1β protein by, for example, competitively modulating aPGC-1β-mediated activity of a PGC-1β protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the PGC-1β protein.

In one embodiment, variants of a PGC-1β protein which function as eitherPGC-1β agonists (mimetics) or as PGC-1β antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a PGC-1β protein for PGC-1β protein agonist or antagonist activity.In one embodiment, a variegated library of PGC-1β variants is generatedby combinatorial mutagenesis at the nucleic acid level and is encoded bya variegated gene library. A variegated library of PGC-1β variants canbe produced by, for example, enzymatically ligating a mixture ofsynthetic oligonucleotides into gene sequences such that a degenerateset of potential PGC-1β sequences is expressible as individualpolypeptides, or alternatively, as a set of larger fusion proteins(e.g., for phage display) containing the set of PGC-1β sequencestherein. There are a variety of methods which can be used to producelibraries of potential PGC-1β variants from a degenerate oligonucleotidesequence. Chemical synthesis of a degenerate gene sequence can beperformed in an automatic DNA synthesizer, and the synthetic gene thenligated into an appropriate expression vector. Use of a degenerate setof genes allows for the provision, in one mixture, of all of thesequences encoding the desired set of potential PGC-1β sequences.Methods for synthesizing degenerate oligonucleotides are known in theart (see, e.g., Narang, S. A. (1983) Tetrahedron 39:3; Itakura et al.(1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science198:1056; Ike et al. (1983) Nucleic Acids Res. 11:477.

In addition, libraries of fragments of a PGC-1β protein coding sequencecan be used to generate a variegated population of PGC-1β fragments forscreening and subsequent selection of variants of a PGC-1β protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of a PGC-1β coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PGC-1β protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PGC-1β proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PGC-1β variants (Arkin and Youvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delagrave et al. (1993) Protein Eng. 6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated PGC-1β library. For example, a library of expression vectorscan be transfected into a cell line, e.g., a brown adipose cell linesuch as HIB1B, which ordinarily responds to a PGC-1β target molecule ina particular PGC-1β target molecule-dependent manner. The transfectedcells are then contacted with a PGC-1β target molecule and the effect ofexpression of the mutant on, e.g., on the transcriptional activity ofPGC-1 P or the target molecule can be detected. Plasmid DNA can then berecovered from the cells which score for inhibition, or alternatively,potentiation of signaling by the PGC-1β target molecule, and theindividual clones further characterized.

An isolated PGC-1β protein, or a portion or fragment thereof, can beused as an immunogen to generate antibodies that bind PGC-1β usingstandard techniques for polyclonal and monoclonal antibody preparation.A full-length PGC-1β protein can be used or, alternatively, theinvention provides antigenic peptide fragments of PGC-1β for use asimmunogens. The antigenic peptide of PGC-1β comprises at least 8 aminoacid residues of the amino acid sequence shown in SEQ ID NO:2 or 5 andencompasses an epitope of PGC-1β such that an antibody raised againstthe peptide forms a specific immune complex with the PGC-1β protein.Preferably, the antigenic peptide comprises at least 10 amino acidresidues, more preferably at least 15 amino acid residues, even morepreferably at least 20 amino acid residues, and most preferably at least30 amino acid residues.

Preferred epitopes encompassed by the antigenic peptide are regions ofPGC-1β that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity.

A PGC-1β immunogen typically is used to prepare antibodies by immunizinga suitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed PGC-1β protein or a chemicallysynthesized PGC-1β polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic PGC-1β preparation induces a polyclonal anti-PGC-1β antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-PGC-1βantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as a PGC-1β.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind PGC-1β molecules. Theterm “monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of PGC-1β. A monoclonal antibody composition thustypically displays a single binding affinity for a particular PGC-1βprotein with which it immunoreacts.

Polyclonal anti-PGC-1β antibodies can be prepared as described above byimmunizing a suitable subject with a PGC-1β immunogen. The anti-PGC-1βantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized PGC-1β. If desired, the antibody moleculesdirected against PGC-1β can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-PGC-1β antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem. 255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol. Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generallyKenneth, R. H. in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); Lerner, E. A.(1981) Yale J. Biol. Med. 54:387-402; Gefter, M. L. et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a PGC-1β immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds PGC-1β.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-PGC-1β monoclonal antibody (see, e.g., Galfre, G. et al. (1977)Nature 266:55052; Gefter et al. (1977) supra; Lerner (1981) supra;Kenneth (1980 supra). Moreover, the ordinarily skilled worker willappreciate that there are many variations of such methods which alsowould be useful. Typically, the immortal cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas can be made by fusing lymphocytes from amouse immunized with an immunogenic preparation of the present inventionwith an immortalized mouse cell line. Preferred immortal cell lines aremouse myeloma cell lines that are sensitive to culture medium containinghypoxanthine, aminopterin and thymidine (“HAT medium”). Any of a numberof myeloma cell lines can be used as a fusion partner according tostandard techniques, e.g., the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 orSp2/O-Ag14 myeloma lines. These myeloma lines are available from ATCC.Typically, HAT-sensitive mouse myeloma cells are fused to mousesplenocytes using polyethylene glycol (“PEG”). Hybridoma cells resultingfrom the fusion are then selected using HAT medium, which kills unfusedand unproductively fused myeloma cells (unfused splenocytes die afterseveral days because they are not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindPGC-1β, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-PGC-1β antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with PGC-1β to thereby isolateimmunoglobulin library members that bind PGC-1β. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrard et al.(1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) NucleicAcids Res. 19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. (1990) Nature 348:552-554.

Additionally, recombinant anti-PGC-1β antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Cancer Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-PGC-1β antibody (e.g., monoclonal antibody) can be used toisolate PGC-1β by standard techniques, such as affinity chromatographyor immunoprecipitation. An anti-PGC-1β antibody can facilitate thepurification of natural PGC-1β from cells and of recombinantly producedPGC-1β expressed in host cells. Moreover, an anti-PGC-1β antibody can beused to detect PGC-1β protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the PGC-1β protein. Anti-PGC-1β antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PGC-1β protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) Methods Enzymol. 185:3-7.Regulatory sequences include those which direct constitutive expressionof a nucleotide sequence in many types of host cells and those whichdirect expression of the nucleotide sequence only in certain host cells(e.g., tissue-specific regulatory sequences). It will be appreciated bythose skilled in the art that the design of the expression vector candepend on such factors as the choice of the host cell to be transformed,the level of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., PGC-1β proteins,mutant forms of PGC-1β proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of PGC-1β proteins in prokaryotic or eukaryotic cells. Forexample, PGC-1β proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel(1990) supra. Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in PGC-1β activity assays,(e.g., direct assays or competitive assays described in detail below),or to generate antibodies specific for PGC-1β proteins, for example. Ina preferred embodiment, a PGC-1β fusion protein expressed in aretroviral expression vector of the present invention can be utilized toinfect bone marrow cells which are subsequently transplanted intoirradiated recipients. The pathology of the subject recipient is thenexamined after sufficient time has passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al. (1990) Methods Enzymol. 185:60-89). Target gene expression fromthe pTrc vector relies on host RNA polymerase transcription from ahybrid trp-lac fusion promoter. Target gene expression from the pET 11dvector relies on transcription from a T7 gn10-lac fusion promotermediated by a coexpressed viral RNA polymerase (T7 gn1). This viralpolymerase is supplied by host strains BL21 (DE3) or HMS174(DE3) from aresident prophage harboring a T7 gn1 gene under the transcriptionalcontrol of the lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S. (1990)Methods Enzymol. 185:119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PGC-1β expression vector is a yeastexpression vector. Examples of vectors for expression in yeast S.cerevisiae include pYepSec1 (Baldari, et al., (1987) EMBO J. 6:229-234),pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz etal., (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (Invitrogen Corp, San Diego, Calif.).

Alternatively, PGC-1β proteins can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf 9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.et al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1 987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol. 43:23 5-275), in particular promoters of Tcell receptors (Winoto and Baltimore (1 989) EMBO J. 8:729-733) andimmunoglobulins (Baneiji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PGC-1β mRNA. Regulatory sequences operativelylinked to a nucleic acid cloned in the antisense orientation can bechosen which direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen which directconstitutive, tissue specific or cell type specific expression of antisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see Weintraub, H. etal., Anti sense RNA as a molecular tool for genetic analysis,Reviews—Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which aPGC-1β nucleic acid molecule of the invention is introduced, e.g., aPGC-1β nucleic acid molecule within a recombinant expression vector or aPGC-1β nucleic acid molecule containing sequences which allow it tohomologously recombine into a specific site of the host cell's genome.The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. It is understood that such terms refer not onlyto the particular subject cell but to the progeny or potential progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aPGC-1β protein can be expressed in bacterial cells such as E. coli,insect cells, yeast or mammalian cells (such as Chinese hamster ovarycells (CHO), COS cells, BOSC cells, or HIB1B cells). Other suitable hostcells are known to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a PGC-1β protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a PGC-1βprotein. Accordingly, the invention further provides methods forproducing a PGC-1β protein using the host cells of the invention. In oneembodiment, the method comprises culturing the host cell of theinvention (into which a recombinant expression vector encoding a PGC-1βprotein has been introduced) in a suitable medium such that a PGC-1βprotein is produced. In another embodiment, the method further comprisesisolating a PGC-1β protein from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichPGC-1β-coding sequences have been introduced. Such host cells can thenbe used to create non-human transgenic animals in which exogenous PGC-1βsequences have been introduced into their genome or homologousrecombinant animals in which endogenous PGC-1β sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a PGC-1β and for identifying and/or evaluating modulators ofPGC-1β activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA which is integrated into the genome of acell from which a transgenic animal develops and which remains in thegenome of the mature animal, thereby directing the expression of anencoded gene product in one or more cell types or tissues of thetransgenic animal. As used herein, a “homologous recombinant animal” isa non-human animal, preferably a mammal, more preferably a mouse, inwhich an endogenous PGC-1β gene has been altered by homologousrecombination between the endogenous gene and an exogenous DNA moleculeintroduced into a cell of the animal, e.g., an embryonic cell of theanimal, prior to development of the animal.

A transgenic animal of the invention can be created by introducing aPGC-1β-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The PGC-1βcDNA sequence of SEQ ID NO:1 or 4 can be introduced as a transgene intothe genome of a non-human animal. Alternatively, a nonhuman homologue ofa human PGC-1β gene, such as a rat PGC-1β gene, can be used as atransgene. Alternatively, a PGC-1β gene homologue, such as anotherPGC-1β family member, can be isolated based on hybridization to thePGC-1β cDNA sequences of SEQ ID NO:1, 3, 4, or 6, (described further insubsection I above) and used as a transgene. Intronic sequences andpolyadenylation signals can also be included in the transgene toincrease the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to aPGC-1β transgene to direct expression of a PGC-1β protein to particularcells. Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a PGC-1β transgene in its genome and/or expression of PGC-1βmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a PGC-1βprotein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a PGC-1β gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PGC-1β gene. The PGC-1β gene can be a humangene (e.g., the cDNA of SEQ ID NO:6), but more preferably, is the mousegene of SEQ ID NO:3, or another non-human homologue of a human PGC-1βgene (e.g., a cDNA isolated by stringent hybridization with thenucleotide sequence of SEQ ID NO:1 or 4). For example, the mouse PGC-1βgene can be used to construct a homologous recombination nucleic acidmolecule, e.g., a vector, suitable for altering an endogenous PGC-1βgene in the mouse genome. In a preferred embodiment, the homologousrecombination nucleic acid molecule is designed such that, uponhomologous recombination, the endogenous PGC-1β gene is functionallydisrupted (i.e., no longer encodes a functional protein; also referredto as a “knock out” vector). Alternatively, the homologous recombinationnucleic acid molecule can be designed such that, upon homologousrecombination, the endogenous PGC-1β gene is mutated or otherwisealtered but still encodes functional protein (e.g., the upstreamregulatory region can be altered to thereby alter the expression of theendogenous PGC-1β protein). In the homologous recombination nucleic acidmolecule, the altered portion of the PGC-1β gene is flanked at its 5′and 3′ ends by additional nucleic acid sequence of the PGC-1β gene toallow for homologous recombination to occur between the exogenous PGC-1βgene carried by the homologous recombination nucleic acid molecule andan endogenous PGC-1β gene in a cell, e.g., an embryonic stem cell. Theadditional flanking PGC-1β nucleic acid sequence is of sufficient lengthfor successful homologous recombination with the endogenous gene.Typically, several kilobases of flanking DNA (both at the 5′ and 3′ends) are included in the homologous recombination nucleic acid molecule(see, e.g., Thomas, K. R. and Capecchi, M. R. (1987) Cell 51:503 for adescription of homologous recombination vectors). The homologousrecombination nucleic acid molecule is introduced into a cell, e.g., anembryonic stem cell line (e.g., by electroporation) and cells in whichthe introduced PGC-1β gene has homologously recombined with theendogenous PGC-1β gene are selected (see e.g., Li, E. et al. (1992) Cell69:915). The selected cells can then injected into a blastocyst of ananimal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley,A. in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach,E. J. Robertson, ed. (IRL, Oxford, 1987) pp. 113-152). A chimeric embryocan then be implanted into a suitable pseudopregnant female fosteranimal and the embryo brought to term. Progeny harboring thehomologously recombined DNA in their germ cells can be used to breedanimals in which all cells of the animal contain the homologouslyrecombined DNA by germline transmission of the transgene. Methods forconstructing homologous recombination nucleic acid molecules, e.g.,vectors, or homologous recombinant animals are described further inBradley, A. (1991) Curr. Opin. Biotechnol. 2:823-829 and in PCTInternational Publication Nos. WO 90/11354 by Le Mouellec et al.; WO91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; and WO93/04169 by Berns et al.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad.Sci. USA 89:6232-6236. Another example of a recombinase system is theFLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al.(1991) Science 251:1351-1355. If a cre/loxP recombinase system is usedto regulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Thereconstructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The PGC-1β nucleic acid molecules, fragments of PGC-1β proteins, PGC-1βmodulators, and anti-PGC-1β antibodies (also referred to herein as“active compounds”) of the invention can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically comprise the nucleic acid molecule, protein, orantibody and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a PGC-1β modulator, a fragment of a PGC-1β protein or ananti-PGC-1 antibody) in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

In certain embodiments of the invention, a modulator of PGC-1β activityis administered in combination with other agents (e.g., a smallmolecule), or in conjunction with another, complementary treatmentregime. For example, in one embodiment, a modulator of PGC-1β activityis used to treat a metabolic disorder. Accordingly, modulation of PGC-1βactivity may be used in conjunction with, for example, another agentused to treat the disorder (e.g., another agent used to treat diabetes,e.g., insulin, metformin, or a thiazoladinedione such as rosiglitizoneor pioglitizone). In another embodiment, a modulator of PGC-1β activityis used to treat a neurodegenerative disorder. Accordingly, modulationof PGC-1β activity may be used in conjunction with, for example, anotheragent used to treat the disorder (e.g., another agent used to treatParkinson's disease, e.g., tolcapone (Tasmar), or another COMT inhibitoror levodopa, levodopa/carbidopa, symmetrel, anticholinergics, selegilineor deprenyl (Eldepryl) or dopamine agonists).

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator;or, biological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, smallmolecules, and antibodies described herein can be used in one or more ofthe following methods: a) screening assays; b) predictive medicine(e.g., diagnostic assays, prognostic assays, monitoring clinical trials,and pharmacogenetics); and c) methods of treatment (e.g., therapeuticand prophylactic). As described herein, a PGC-1β protein of theinvention has one or more of the following activities: 1) it interactswith a nuclear receptor (e.g., HNF4α, PPARα, retinoic acid receptor α(RARα), thyroid hormone receptor β (TRβ), or glucocorticoid receptor(GR)); 2) it interacts with HCF; 3) it interacts with NRF1; 4) itinteracts with a basal transcription factor; 5) it modulates theactivity, e.g., the transcriptional activity, of a nuclear receptorand/or NRF1; 6) it modulates brown adipose cell determination and/ordifferentiation; 7) it modulates intra- or inter-cellular signaling; 8)it modulates viral infection (e.g., via interaction with HCF); 9) itmodulates cellular proliferation; 10) it modulates metabolism; 11) itmodulates mitochondrial activity and/or biogenesis; and 12) it modulatesfatty acid β-oxidation.

The isolated nucleic acid molecules of the invention can be used, forexample, to express PGC-1β protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect PGC-1βmRNA (e.g., in a biological sample) or a genetic alteration in a PGC-1βgene, and to modulate PGC-1β activity, as described further below. ThePGC-1β proteins can be used to treat disorders characterized byinsufficient or excessive production of a PGC-1β target molecule orproduction of PGC-1β inhibitors. In addition, the PGC-1β proteins can beused to screen for naturally occurring PGC-1β target molecules, toscreen for drugs or compounds which modulate PGC-1β activity, as well asto treat disorders characterized by insufficient or excessive productionof PGC-1β protein or production of PGC-1β protein forms which havedecreased, aberrant or unwanted activity compared to PGC-1β wild typeprotein (e.g., metabolic disorders, such as diabetes, insulinresistance, obesity, overweight, anorexia, and cachexia; cellular growthor differentiation disorders; and viral disorders). Moreover, theanti-PGC-1β antibodies of the invention can be used to detect andisolate PGC-1β proteins, regulate the bioavailability of PGC-1βproteins, and modulate PGC-1β activity.

A. Screening Assays:

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, e.g., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to PGC-1β proteins, have a stimulatory or inhibitory effecton, for example, PGC-1β expression or PGC-1β activity, or have astimulatory or inhibitory effect on, for example, the expression oractivity of a PGC-1β target molecule. The invention further provides amethod (also referred to herein as a “screening assay”) for identifyingmodulators, e.g., candidate or test compounds or agents (e.g., peptides,peptidomimetics, small molecules or other drugs) which modulate, e.g.,upregulate or downregulated, the interaction between PGC-1α and HCF viathe HCF binding motif (HBM). Those of skill in the art will appreciatethat any of the following methods may be used to identify compoundswhich modulate the PGC-1α-HCF interaction in order to identify compoundswhich modulate cellular proliferation and/or viral infection. Thenucleotide and amino acid sequences of mouse PGC-1α are set forth in SEQID NOs:8 and 9, respectively, and are described in U.S. Pat. No.6,166,192; PCT International Publication No. WO 00/32215; Puigserver, P.et al. (1998) Cell 92(6):829-39, the contents of all of which areincorporated herein by reference. The nucleotide and amino acidsequences of human PGC-1α are set forth in SEQ ID NOs:10 and 111,respectively, and are described in PCT International Publication No. WO00/32215.

In one embodiment, the invention provides assays for screening candidateor test compounds which are target molecules of a PGC-1β protein orpolypeptide or biologically active portion thereof (e.g., nuclearreceptors or other transcription factors). In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a PGC-1β protein orpolypeptide or biologically active portion thereof (e.g., cofactor orcoenzyme analogs, or inhibitory molecules). The l0 test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. USA 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a PGC-1β protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate PGC-1β activity is determined. Determining the ability of thetest compound to modulate PGC-1β activity can be accomplished bymonitoring, for example, the interaction with and/or coactivation of aknown target molecule (e.g., a nuclear receptor or HCF), by monitoringthe autonomous transcriptional activity of PGC-1β, by monitoring theproduction of one or more specific metabolites in a cell which expressesPGC-1β (e.g., ¹⁴C glucose), by monitoring expression of mitochondrialgenes, or by monitoring mitochondrial content or function in the cell.The cell, for example, can be of mammalian origin, e.g., a brown adiposecell such as a HIB1B cell, a heart cell, or a liver cell.

The ability of the test compound to modulate PGC-1β binding to a targetmolecule (e.g., a nuclear receptor or HCF) can also be determined.Determining the ability of the test compound to modulate PGC-1β bindingto a target molecule can be accomplished, for example, by coupling thePGC-1β target molecule with a radioisotope or enzymatic label such thatbinding of the PGC-1β target molecule to PGC-1β can be determined bydetecting the labeled PGC-1β target molecule in a complex.Alternatively, PGC-1β could be coupled with a radioisotope or enzymaticlabel to monitor the ability of a test compound to modulate PGC-1βbinding to a PGC-1β target molecule in a complex. Determining theability of the test compound to bind PGC-1β can be accomplished, forexample, by coupling the compound with a radioisotope or enzymatic labelsuch that binding of the compound to PGC-1β can be determined bydetecting the labeled PGC-1β compound in a complex. For example,compounds (e.g., PGC-1β target molecule, including small molecules) canbe labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly,and the radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., a PGC-1β target molecule) to interact with PGC-1βwithout the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interaction of a compoundwith PGC-1β without the labeling of either the compound or the PGC-1β.McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a“microphysiometer” (e.g., Cytosensor) is an analytical instrument thatmeasures the rate at which a cell acidifies its environment using alight-addressable potentiometric sensor (LAPS). Changes in thisacidification rate can be used as an indicator of the interactionbetween a compound and PGC-1β.

The ability of a test compound to modulate PGC-1β activity can bemeasured by contacting a cell, e.g., an undifferentiated HIB1B cell, anddetermining the ability of the compound to modulate differentiation ofthe cell into a brown adipose cell. The ability of a test compound tomodulate PGC-1β activity can also be measured by contacting a cell(e.g., a brown adipose cell) with the test compound and measuring thenumber of mitochondria or the level of mitochondrial function in thecell as compared to a control cell not contacted with the test compound.The number of mitochondria can be measured, for example, by counting themitochondria present in electron microscopy sections of the cell, or byanalyzing the amount of mitochondrial DNA present in the cell, forexample, by Southern blotting. Mitochondrial function can be determinedby measuring expression levels of mitochondrial genes such as cytochromec oxidase or by measuring oxygen consumption by the cell.

Exemplary methods for measuring mitochondrial function can further befound in: U.S. Pat. No. 6,166,192; PCT International Publication No. WO00/32215; Puigserver, P. et al. (1998) Cell 92(6):829-39; Vidal-Puig, A.J. et al. (2000) J. Biol. Chem. 275(21):16258-66; and Wu, Z. et al.(1999) Cell 98(l):115-24, the entire contents of all of which areincorporated herein by reference.

The ability of a test compound to modulate insulin sensitivity of a cellcan be determined by performing an assay in which a cell which expressPGC-1β, e.g., a brown adipose cell such as a HIB1B cell, is contactedwith the test compound, e.g., transformed to express the test compound;incubated with radioactively labeled glucose (¹⁴C glucose); and treatedwith insulin. An increase or decrease in glucose in the cells containingthe test compound as compared to control cells indicates that the testcompound can modulate insulin sensitivity of the cells. Alternatively,the cells containing the test compound can be incubated with aradioactively labeled phosphate source (e.g., [³²P]ATP) and treated withinsulin. Phosphorylation of proteins in the insulin pathway, e.g., theinsulin receptor, can then be measured. An increase or decrease inphosphorylation of a protein in the insulin pathway in cells containingthe test compound as compared to the control cells indicates that thetest compound can modulate insulin sensitivity of the cells.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a PGC-1β target molecule (e.g., a nuclearreceptor or HCF) with a test compound and determining the ability of thetest compound to modulate (e.g., stimulate or inhibit) the activity ofthe PGC-1β target molecule. Determining the ability of the test compoundto modulate the activity of a PGC-1β target molecule can beaccomplished, for example, by determining the ability of the PGC-1βprotein to bind to or interact with the PGC-1β target molecule.Determining the ability of the test compound to modulate the activity ofa PGC-1β target molecule can be accomplished, for example, bydetermining the transcriptional activity of the PGC-1β target molecule.

Determining the ability of the PGC-1β protein, or a biologically activefragment thereof, to bind to or interact with a PGC-1β target moleculecan be accomplished by one of the methods described above fordetermining direct binding. In a preferred embodiment, determining theability of the PGC-1β protein to bind to or interact with a PGC-1βtarget molecule can be accomplished by determining the activity of thetarget molecule. For example, the activity of the target molecule can bedetermined by detecting induction of a cellular response (i.e., changesin cellular proliferation, mitochondrial activity or content, orgluconeogenesis), detecting catalytic/enzymatic activity of the targeton an appropriate substrate, detecting the induction of a reporter gene(comprising a target-responsive regulatory element operatively linked toa nucleic acid encoding a detectable marker, e.g., luciferase), ordetecting a target-regulated cellular response.

In yet another embodiment, an assay of the present invention is acell-free assay in which a PGC-1β protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the PGC-1β protein or biologically active portionthereof is determined. Preferred biologically active portions of thePGC-1β proteins to be used in assays of the present invention includefragments which participate in interactions with non-PGC-1β molecules,e.g., fragments with high surface probability scores. Binding of thetest compound to the PGC-1β protein can be determined either directly orindirectly as described above. In a preferred embodiment, the assayincludes contacting the PGC-1β protein or biologically active portionthereof with a known compound which binds PGC-1β to form an assaymixture, contacting the assay mixture with a test compound, anddetermining the ability of the test compound to interact with a PGC-1βprotein, wherein determining the ability of the test compound tointeract with a PGC-1β protein comprises determining the ability of thetest compound to preferentially bind to PGC-1β or biologically activeportion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a PGC-1βprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the PGC-1β protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a PGC-1β protein can beaccomplished, for example, by determining the ability of the PGC-1βprotein to bind to a PGC-1β target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the PGC-1β protein to bind to a PGC-1β target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a PGC-1β protein can beaccomplished by determining the ability of the PGC-1β protein to furthermodulate the activity of a downstream effector of a PGC-1β targetmolecule. For example, the activity of the effector molecule on anappropriate target can be determined or the binding of the effector toan appropriate target can be determined as previously described.

In yet another embodiment, the cell-free assay involves contacting aPGC-1β protein or biologically active portion thereof with a knowncompound which binds the PGC-1β protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the PGC-1β protein,wherein determining the ability of the test compound to interact withthe PGC-1β protein comprises determining the ability of the PGC-1βprotein to preferentially bind to or catalyze the transfer of a hydridemoiety to or from the target substrate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either PGC-1β or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a PGC-1β protein, or interaction ofa PGC-1β protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/PGC-1β fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or PGC-1β protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level ofPGC-1β binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a PGC-1βprotein or a PGC-1β target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated PGC-1β protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with PGC-1β protein or target molecules but which donot interfere with binding of the PGC-1β protein to its target moleculecan be derivatized to the wells of the plate, and unbound target orPGC-1β protein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the PGC-1β protein or target molecule, as wellas enzyme-linked assays which rely on detecting an enzymatic activityassociated with the PGC-1β protein or target molecule.

In another embodiment, modulators of PGC-1β expression are identified ina method wherein a cell is contacted with a candidate compound and theexpression of PGC-1β mRNA or protein in the cell is determined. Thelevel of expression of PGC-1β mRNA or protein in the presence of thecandidate compound is compared to the level of expression of PGC-1β mRNAor protein in the absence of the candidate compound. The candidatecompound can then be identified as a modulator of PGC-1β expressionbased on this comparison. For example, when expression of PGC-1β mRNA orprotein is greater (statistically significantly greater) in the presenceof the candidate compound than in its absence, the candidate compound isidentified as a stimulator of PGC-1β mRNA or protein expression.Alternatively, when expression of PGC-1β mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of PGC-1β mRNA or protein expression. The level of PGC-1β mRNAor protein expression in the cells can be determined by methodsdescribed herein for detecting PGC-1β mRNA or protein.

In yet another aspect of the invention, the PGC-1β proteins can be usedas “bait proteins” in a two-hybrid assay or three-hybrid assay (see,e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232;Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al.(1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with PGC-1β (“PGC-1β-binding proteins” or“PGC-1β-bp”) and are involved in PGC-1β activity. Such PGC-1β-bindingproteins are also likely to be involved in the propagation of signals bythe PGC-1β proteins or PGC-1β targets as, for example, downstreamelements of a PGC-1β-mediated signaling pathway. Alternatively, suchPGC-1β-binding proteins are likely to be PGC-1β inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a PGC-1β proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aPGC-1β-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the PGC-1β protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a PGC-1β protein can beconfirmed in vivo, e.g., in an animal such as an animal model for in ananimal such as an animal model for obesity, diabetes, anorexia, orcachexia. Examples of animals that can be used include the transgenicmouse described in U.S. Pat. No. 5,932,779 that contains a mutation inan endogenous melanocortin-4-receptor (MC4-R) gene; animals havingmutations which lead to syndromes that include obesity symptoms(described in, for example, Friedman, J. M. et al. (1991) Mamm. Genome1:130-144; Friedman, J. M. and Liebel, R. L. (1992) Cell 69:217-220;Bray, G. A. (1992) Prog. Brain Res. 93:333-341; and Bray, G. A. (1989)Amer. J. Clin. Nutr. 5:891-902); the animals described in Stubdal H. etal. (2000) Mol. Cell Biol. 20(3):878-82 (the mouse tubby phenotypecharacterized by maturity-onset obesity); the animals described inAbadie J. M. et al. Lipids (2000) 35(6):613-20 (the obese Zucker rat(ZR), a genetic model of human youth-onset obesity and type 2 diabetesmellitus); the animals described in Shaughnessy S. et al. (2000)Diabetes 49(6):904-11 (mice null for the adipocyte fatty acid bindingprotein); or the animals described in Loskutoff D. J. et al. (2000) Ann.N. Y Acad. Sci. 902:272-81 (the fat mouse). Other examples of animalsthat may be used include non-recombinant, non-genetic animal models ofobesity such as, for example, rabbit, mouse, or rat models in which theanimal has been exposed to either prolonged cold or long-termover-eating, thereby, inducing hypertrophy of BAT and increasing BATthermogenesis (Himms-Hagen, J. (1990), supra). Additionally, animalscreated by ablation of BAT through use of targeted expression of a toxingene (Lowell, B. et al. (1993) Nature 366:740-742) may be used. Animalsdeficient in PGC-1 (e.g., PGC-1 knockout mice) may be deficient in theability to induce thermogenesis and therefore may be useful indetermining whether a test compound can induce thermogenesis bybypassing PGC-1 and directly modulating the activity of DHDR-2.

In another embodiment of the invention, the ability of the agent tomodulate the activity of a PGC-1β protein can be tested in an animalsuch as an animal model for a cellular proliferation disorder, e.g.,tumorigenesis. Animal based models for studying tumorigenesis in vivoare well known in the art (reviewed in Animal Models of CancerPredisposition Syndromes, Hiai, H. and Hino, O. (eds.) 1999, Progress inExperimental Tumor Research, Vol. 35; Clarke, A. R. (2000)Carcinogenesis 21:435-41) and include, for example, carcinogen-inducedtumors (Rithidech, K. et al. (1999) Mutat. Res. 428:33-39; Miller, M. L.et al. (2000) Environ. Mol. Mutagen. 35:319-327), injection and/ortransplantation of tumor cells into an animal, as well as animalsbearing mutations in growth regulatory genes, for example, oncogenes(e.g., ras) (Arbeit, J. M. et al. (1993) Am. J. Pathol. 142:1187-1197;Sinn, E. et al. (1987) Cell 49:465-475; Thorgeirsson, S S et al. ToxicolLett (2000) 112-113:553-555) and tumor suppressor genes (e.g., p53)(Vooijs, M. et al. (1999) Oncogene 18:5293-5303; Clark A. R. (1995)Cancer Metast. Rev. 14:125-148; Kumar, T. R. et al. (1995) J. Intern.Med. 238:233-238; Donehower, L. A. et al. (1992) Nature 356215-221).Furthermore, experimental model systems are available for the study of,for example, ovarian cancer (Hamilton, T. C. et al. (1984) Semin. Oncol.11:285-298; Rahman, N. A. et al. (1998) Mol. Cell. Endocrinol.145:167-174; Beamer, W. G. et al. (1998) Toxicol. Pathol. 26:704-710),gastric cancer (Thompson, J. et al. (2000) Int. J. Cancer 86:863-869;Fodde, R. et al. (1999) Cytogenet. Cell Genet. 86:105-111), breastcancer (Li, M. et al. (2000) Oncogene 19:1010-1019; Green, J. E. et al.(2000) Oncogene 19:1020-1027), melanoma (Satyamoorthy, K. et al. (1999)Cancer Metast. Rev. 18:401-405), and prostate cancer (Shirai, T. et al.(2000) Mutat. Res. 462:219-226; Bostwick, D. G. et al. (2000) Prostate43:286-294). Animal based models for studying Parkinson's disease arealso well known in the art (Dawson et al., Neuron. Jul. 18,2002;35(2):219-22)

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model, as described above. For example, an agentidentified as described herein (e.g., a PGC-1β modulating agent, anantisense PGC-1β nucleic acid molecule, a PGC-1β-specific antibody, or aPGC-1β-binding partner) can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments as describedherein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the PGC-1β nucleotide sequences, describedherein, can be used to map the location of the PGC-1β genes on achromosome. The mapping of the PGC-1β sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, PGC-1β genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the PGC-1β nucleotidesequences. Computer analysis of the PGC-1β sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the PGC-1β sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the PGC-1βnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a PGC-1βsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data (such data are found, for example, in McKusick,V., Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the PGC-1β gene can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The PGC-1β sequences of the present invention can also be used toidentify individuals from minute biological samples. The United Statesmilitary, for example, is considering the use of restriction fragmentlength polymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the PGC-1β nucleotide sequences described herein can beused to prepare two PCR primers from the 5′ and 3′ ends of thesequences. These primers can then be used to amplify an individual's DNAand subsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The PGC-1β nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. The noncoding sequences of SEQ ID NO:1 or 4can comfortably provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:3 or 6 are used, a more appropriate number of primers forpositive individual identification would be 500-2,000.

If a panel of reagents from PGC-1β nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of PGC-1β Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e., another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of SEQ ID NO:1 or 4 are particularlyappropriate for this use as greater numbers of polymorphisms occur inthe noncoding regions, making it easier to differentiate individualsusing this technique. Examples of polynucleotide reagents include thePGC-1β nucleotide sequences or portions thereof, e.g., fragments derivedfrom the noncoding regions of SEQ ID NO:1 or 4 having a length of atleast 20 bases, preferably at least 30 bases.

The PGC-1β nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., thymus or brain tissue. This can bevery useful in cases where a forensic pathologist is presented with atissue of unknown origin. Panels of such PGC-1β probes can be used toidentify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., PGC-1β primers or probes canbe used to screen tissue culture for contamination (i.e., screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine:

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining PGC-1β proteinand/or nucleic acid expression as well as PGC-1β activity, in thecontext of a biological sample (e.g., blood, serum, cells, ascites,tissue) to thereby determine whether an individual is afflicted with adisease or disorder, or is at risk of developing a disorder, associatedwith aberrant or unwanted PGC-1β expression or activity (e.g., ametabolic disorder or a cellular proliferation disorder). The inventionalso provides for prognostic (or predictive) assays for determiningwhether an individual is at risk of developing a disorder associatedwith PGC-1β protein, nucleic acid expression or activity. For example,mutations in a PGC-1β gene can be assayed in a biological sample. Suchassays can be used for prognostic or predictive purpose to therebyprophylactically treat an individual prior to the onset of a disordercharacterized by or associated with PGC-1β protein, nucleic acidexpression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of PGC-1βin clinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of PGC-1βprotein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting PGC-1β proteinor nucleic acid (e.g., mRNA, or genomic DNA) that encodes PGC-1β proteinsuch that the presence of PGC-1β protein or nucleic acid is detected inthe biological sample. A preferred agent for detecting PGC-1β mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing toPGC-1β mRNA or genomic DNA. The nucleic acid probe can be, for example,the PGC-1β nucleic acid set forth in SEQ ID NO:1, 3, 4, or 6, or aportion thereof, such as an oligonucleotide of at least 15, 30, 50, 100,250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to PGC-1β mRNA or genomic DNA.Other suitable probes for use in the diagnostic assays of the inventionare described herein.

A preferred agent for detecting PGC-1β protein is an antibody capable ofbinding to PGC-1β protein, preferably an antibody with a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can beused. The term “labeled”, with regard to the probe or antibody, isintended to encompass direct labeling of the probe or antibody bycoupling (i.e., physically linking) a detectable substance to the probeor antibody, as well as indirect labeling of the probe or antibody byreactivity with another reagent that is directly labeled. Examples ofindirect labeling include detection of a primary antibody using afluorescently labeled secondary antibody and end-labeling of a DNA probewith biotin such that it can be detected with fluorescently labeledstreptavidin. The term “biological sample” is intended to includetissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject. That is, thedetection method of the invention can be used to detect PGC-1β mRNA,protein, or genomic DNA in a biological sample in vitro as well as invivo. For example, in vitro techniques for detection of PGC-1β mRNAinclude Northern hybridizations and in situ hybridizations. In vitrotechniques for detection of PGC-1β protein include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence. In vitro techniques for detection of PGC-1β genomicDNA include Southern hybridizations. Furthermore, in vivo techniques fordetection of PGC-1β protein include introducing into a subject a labeledanti-PGC-1β antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting PGC-1β protein, mRNA, orgenomic DNA, such that the presence of PGC-1β protein, mRNA or genomicDNA is detected in the biological sample, and comparing the presence ofPGC-1β protein, mRNA or genomic DNA in the control sample with thepresence of PGC-1β protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of PGC-1βin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting PGC-1β protein or mRNA in abiological sample; means for determining the amount of PGC-1β in thesample; and means for comparing the amount of PGC-1β in the sample witha standard. The compound or agent can be packaged in a suitablecontainer. The kit can further comprise instructions for using the kitto detect PGC-1β protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant or unwanted PGC-1β expression or activity(e.g., a metabolic disorder or a cellular proliferation disorder). Asused herein, the term “aberrant” includes a PGC-1β expression oractivity which deviates from the wild type PGC-1β expression oractivity. Aberrant expression or activity includes increased ordecreased expression or activity, as well as expression or activitywhich does not follow the wild type developmental pattern of expressionor the subcellular pattern of expression. For example, aberrant PGC-1βexpression or activity is intended to include the cases in which amutation in the PGC-1β gene causes the PGC-1β gene to be under-expressedor over-expressed and situations in which such mutations result in anon-functional PGC-1β protein or a protein which does not function in awild-type fashion, e.g., a protein which does not interact with a PGC-1βtarget molecule, or one which interacts with a non-PGC-1β targetmolecule. As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response such as cellularproliferation. For example, the term unwanted includes a PGC-1βexpression or activity which is undesirable in a subject.

The assays described herein, such as the preceding diagnostic assays orthe following assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in PGC-1βprotein activity or nucleic acid expression, such as a metabolicdisorder or a cellular proliferation disorder. Alternatively, theprognostic assays can be utilized to identify a subject having or atrisk for developing a disorder associated with a misregulation in PGC-1βprotein activity or nucleic acid expression, such as a metabolicdisorder or a cellular proliferation disorder. Thus, the presentinvention provides a method for identifying a disease or disorderassociated with aberrant or unwanted PGC-1β expression or activity inwhich a test sample is obtained from a subject and PGC-1β protein ornucleic acid (e.g., mRNA or genomic DNA) is detected, wherein thepresence of PGC-1β protein or nucleic acid is diagnostic for a subjecthaving or at risk of developing a disease or disorder associated withaberrant or unwanted PGC-1β expression or activity. As used herein, a“test sample” refers to a biological sample obtained from a subject ofinterest. For example, a test sample can be a biological fluid (e.g.,blood, ascites, cerebrospinal fluid, or serum), cell sample, or tissuesample (e.g., a fat, heart, or liver sample).

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant or unwanted PGC-1β expression or activity. Forexample, such methods can be used to determine whether a subject can beeffectively treated with an agent for a metabolic disorder or a cellularproliferation disorder. Thus, the present invention provides methods fordetermining whether a subject can be effectively treated with an agentfor a disorder associated with aberrant or unwanted PGC-1β expression oractivity in which a test sample is obtained and PGC-1β protein ornucleic acid expression or activity is detected (e.g., wherein theabundance of PGC-1β protein or nucleic acid expression or activity isdiagnostic for a subject that can be administered the agent to treat adisorder associated with aberrant or unwanted PGC-1β expression oractivity).

The methods of the invention can also be used to detect geneticalterations in a PGC-1β gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inPGC-1β protein activity or nucleic acid expression, such as a metabolicdisorder or a cellular proliferation disorder. In preferred embodiments,the methods include detecting, in a sample of cells from the subject,the presence or absence of a genetic alteration characterized by atleast one of an alteration affecting the integrity of a gene encoding aPGC-1β-protein, or the mis-expression of the PGC-1β gene. For example,such genetic alterations can be detected by ascertaining the existenceof at least one of 1) a deletion of one or more nucleotides from aPGC-1β gene; 2) an addition of one or more nucleotides to a PGC-1β gene;3) a substitution of one or more nucleotides of a PGC-1β gene, 4) achromosomal rearrangement of a PGC-1β gene; 5) an alteration in thelevel of a messenger RNA transcript of a PGC-1β gene, 6) aberrantmodification of a PGC-1β gene, such as of the methylation pattern of thegenomic DNA, 7) the presence of a non-wild type splicing pattern of amessenger RNA transcript of a PGC-1β gene, 8) a non-wild type level of aPGC-1β-protein, 9) allelic loss of a PGC-1β gene, and 10) inappropriatepost-translational modification of a PGC-1β-protein. As describedherein, there are a large number of assays known in the art which can beused for detecting alterations in a PGC-1β gene. A preferred biologicalsample is a tissue (e.g., a fat, heart, or liver sample), blood, orserum sample isolated by conventional means from a subject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in a PGC-1β gene (seeAbravaya et al. (1995) Nucleic Acids Res. 23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a PGC-1β gene under conditions such thathybridization and amplification of the PGC-1β gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a PGC-1β gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in PGC-1β can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Hum. Mutat. 7:244-255; Kozal, M. J.et al. (1996) Nat. Med. 2:753-759). For example, genetic mutations inPGC-1β can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the PGC-1β gene anddetect mutations by comparing the sequence of the sample PGC-1β with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the PGC-1β gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type PGC-1β sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl. Acad. Sci. USA 85:4397; Saleeba et al. (1992) MethodsEnzymol. 217:286-295. In a preferred embodiment, the control DNA or RNAcan be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in PGC-1β cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a PGC-1βsequence, e.g., a wild-type PGC-1β sequence, is hybridized to a cDNA orother DNA product from a test cell(s). The duplex is treated with a DNAmismatch repair enzyme, and the cleavage products, if any, can bedetected from electrophoresis protocols or the like. See, for example,U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in PGC-1β genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA:86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and controlPGC-1β nucleic acids will be denatured and allowed to renature. Thesecondary structure of single-stranded nucleic acids varies according tosequence, the resulting alteration in electrophoretic mobility enablesthe detection of even a single base change. The DNA fragments may belabeled or detected with labeled probes. The sensitivity of the assaymay be enhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet. 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl. Acad. Sci.USA 86:6230). Such allele specific oligonucleotides are hybridized toPCR amplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site in l0the region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a PGC-1β gene.

Furthermore, any cell type or tissue in which PGC-1β is expressed may beutilized in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a PGC-1β protein (e.g., the modulation of brown adiposedifferentiation, metabolism, and/or cellular proliferation) can beapplied not only in basic drug screening, but also in clinical trials.For example, the effectiveness of an agent determined by a screeningassay as described herein to increase PGC-1β gene expression, proteinlevels, or upregulate PGC-1β activity, can be monitored in clinicaltrials of subjects exhibiting decreased PGC-1β gene expression, proteinlevels, or downregulated PGC-1β activity. Alternatively, theeffectiveness of an agent determined by a screening assay to decreasePGC-1β gene expression, protein levels, or downregulate PGC-1β activity,can be monitored in clinical trials of subjects exhibiting increasedPGC-1β gene expression, protein levels, or upregulated PGC-1β activity.In such clinical trials, the expression or activity of a PGC-1β gene,and preferably, other genes that have been implicated in, for example, aPGC-1β-associated disorder can be used as a “read out” or markers of thephenotype of a particular cell.

For example, and not by way of limitation, genes, including PGC-1β, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates PGC-1β activity (e.g., identified ina screening assay as described herein) can be identified. Thus, to studythe effect of agents on PGC-1β-associated disorders (e.g., disorderscharacterized by deregulated brown adipose differentiation,gluconeogenesis, and/or cell proliferation), for example, in a clinicaltrial, cells can be isolated and RNA prepared and analyzed for thelevels of expression of PGC-1β and other genes implicated in thePGC-1β-associated disorder, respectively. The levels of gene expression(e.g., a gene expression pattern) can be quantified by northern blotanalysis or RT-PCR, as described herein, or alternatively by measuringthe amount of protein produced, by one of the methods as describedherein, or by measuring the levels of activity of PGC-1β or other genes.In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent.Accordingly, this response state may be determined before, and atvarious points during treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a PGC-1β protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the PGC-1β protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the PGC-1β protein, mRNA, or genomic DNA inthe pre-administration sample with the PGC-1β protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of PGC-1β to higher levels than detected, i.e.,to increase the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of PGC-1β to lower levels than detected, i.e., to decrease theeffectiveness of the agent. According to such an embodiment, PGC-1βexpression or activity may be used as an indicator of the effectivenessof an agent, even in the absence of an observable phenotypic response.

D. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted PGC-1βexpression or activity, e.g., a metabolic disorder or a cellularproliferation disorder. As used herein, “treatment” of a subjectincludes the application or administration of a therapeutic agent to asubject, or application or administration of a therapeutic agent to acell or tissue from a subject, who has a diseases or disorder, has asymptom of a disease or disorder, or is at risk of (or susceptible to) adisease or disorder, with the purpose to cure, heal, alleviate, relieve,alter, remedy, ameliorate, improve, or affect the disease or disorder,the symptom of the disease or disorder, or the risk of (orsusceptibility to) the disease or disorder. As used herein, a“therapeutic agent” includes, but is not limited to, small molecules,peptides, polypeptides, antibodies, ribozymes, and antisenseoligonucleotides.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the PGC-1β moleculesof the present invention or PGC-1β modulators according to thatindividual's drug response genotype. Pharmacogenomics allows a clinicianor physician to target prophylactic or therapeutic treatments topatients who will most benefit from the treatment and to avoid treatmentof patients who will experience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedPGC-1β expression or activity, by administering to the subject a PGC-1βor an agent which modulates PGC-1β expression or at least one PGC-1βactivity. Subjects at risk for a disease which is caused or contributedto by aberrant or unwanted PGC-1β expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe PGC-1β aberrancy, such that a disease or disorder is prevented or,alternatively, delayed in its progression. Depending on the type ofPGC-1β aberrancy, for example, a PGC-1β, PGC-1β agonist or PGC-1βantagonist agent can be used for treating the subject. The appropriateagent can be determined based on screening assays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating PGC-1βexpression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a PGC-1β or agent that modulates one or more ofthe activities of PGC-1β protein activity associated with the cell. Anagent that modulates PGC-1β protein activity can be an agent asdescribed herein, such as a nucleic acid or a protein, anaturally-occurring target molecule of a PGC-1β protein (e.g., a nuclearreceptor or HCF), a PGC-1β antibody, a PGC-1β agonist or antagonist, apeptidomimetic of a PGC-1β agonist or antagonist, or other smallmolecule. In one embodiment, the agent stimulates one or more PGC-1βactivities. Examples of such stimulatory agents include active PGC-1βprotein and a nucleic acid molecule encoding PGC-1β that has beenintroduced into the cell. In another embodiment, the agent inhibits oneor more PGC-1β activities. Examples of such inhibitory agents includeantisense PGC-1β nucleic acid molecules, anti-PGC-1β antibodies, andPGC-1β inhibitors. These modulatory methods can be performed in vitro(e.g., by culturing the cell with the agent) or, alternatively, in vivo(e.g., by administering the agent to a subject). As such, the presentinvention provides methods of treating an individual afflicted with adisease or disorder characterized by aberrant or unwanted expression oractivity of a PGC-1β protein or nucleic acid molecule. In oneembodiment, the method involves administering an agent (e.g., an agentidentified by a screening assay described herein), or combination ofagents that modulates (e.g., upregulates or downregulates) PGC-1βexpression or activity. In another embodiment, the method involvesadministering a PGC-1β protein or nucleic acid molecule as therapy tocompensate for reduced, aberrant, or unwanted PGC-1β expression oractivity.

Stimulation of PGC-1β activity is desirable in situations in whichPGC-1β is abnormally downregulated and/or in which increased PGC-1βactivity is likely to have a beneficial effect. Likewise, inhibition ofPGC-1β activity is desirable in situations in which PGC-1β is abnormallyupregulated and/or in which decreased PGC-1β activity is likely to havea beneficial effect.

3. Pharmacogenomics

The PGC-1β molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on PGC-1βactivity (e.g., PGC-1β gene expression) as identified by a screeningassay described herein can be administered to individuals to treat(prophylactically or therapeutically) PGC-1β-associated disorders (e.g.,metabolic disorders or a cellular proliferation disorders) associatedwith aberrant or unwanted PGC-1β activity. In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a PGC-1β molecule or PGC-1βmodulator as well as tailoring the dosage and/or therapeutic regimen oftreatment with a PGC-1β molecule or PGC-1β modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants). Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a PGC-1βprotein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling” can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a PGC-1β moleculeor PGC-1β modulator of the present invention) can give an indicationwhether gene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a PGC-1β moleculeor PGC-1β modulator, such as a modulator identified by one of theexemplary screening assays described herein.

E. Electronic Apparatus Readable Media and Arrays

Electronic apparatus readable media comprising PGC-1β sequenceinformation is also provided. As used herein, “PGC-1β sequenceinformation” refers to any nucleotide and/or amino acid sequenceinformation particular to the PGC-1β molecules of the present invention,including but not limited to full-length nucleotide and/or amino acidsequences, partial nucleotide and/or amino acid sequences, polymorphicsequences including single nucleotide polymorphisms (SNPs), epitopesequences, and the like. Moreover, information “related to” said PGC-1βsequence information includes detection of the presence or absence of asequence (e.g., detection of expression of a sequence, fragment,polymorphism, etc.), determination of the level of a sequence (e.g.,detection of a level of expression, for example, a quantitativedetection), detection of a reactivity to a sequence (e.g., detection ofprotein expression and/or levels, for example, using a sequence-specificantibody), and the like. As used herein, “electronic apparatus readablemedia” refers to any suitable medium for storing, holding, or containingdata or information that can be read and accessed directly by anelectronic apparatus. Such media can include, but are not limited to:magnetic storage media, such as floppy discs, hard disc storage medium,and magnetic tape; optical storage media such as compact discs;electronic storage media such as RAM, ROM, EPROM, EEPROM and the like;and general hard disks and hybrids of these categories such asmagnetic/optical storage media. The medium is adapted or configured forhaving recorded thereon PGC-1β sequence information of the presentinvention.

As used herein, the term “electronic apparatus” is intended to includeany suitable computing or processing apparatus or other deviceconfigured or adapted for storing data or information. Examples ofelectronic apparatus suitable for use with the present invention includestand-alone computing apparatuses; networks, including a local areanetwork (LAN), a wide area network (WAN) Internet, Intranet, andExtranet; electronic appliances such as a personal digital assistants(PDAs), cellular phone, pager and the like; and local and distributedprocessing systems.

As used herein, “recorded” refers to a process for storing or encodinginformation on the electronic apparatus readable medium. Those skilledin the art can readily adopt any of the presently known methods forrecording information on known media to generate manufactures comprisingthe PGC-1β sequence information.

A variety of software programs and formats can be used to store thesequence information on the electronic apparatus readable medium. Forexample, the sequence information can be represented in a wordprocessing text file, formatted in commercially-available software suchas WordPerfect and Microsoft Word, represented in the form of an ASCIIfile, or stored in a database application, such as DB2, Sybase, Oracle,or the like, as well as in other forms. Any number of dataprocessorstructuring formats (e.g., text file or database) may be employed inorder to obtain or create a medium having recorded thereon the PGC-1βsequence information.

By providing PGC-1β sequence information in readable form, one canroutinely access the sequence information for a variety of purposes. Forexample, one skilled in the art can use the sequence information inreadable form to compare a target sequence or target structural motifwith the sequence information stored within the data storage means.Search means are used to identify fragments or regions of the sequencesof the invention which match a particular target sequence or targetmotif.

The present invention therefore provides a medium for holdinginstructions for performing a method for determining whether a subjecthas a metabolic or cellular proliferation disease, disorder, orpre-disease condition or a pre-disposition to a metabolic or cellularproliferation disease, disorder, or pre-disease condition, wherein themethod comprises the steps of determining PGC-1β sequence informationassociated with the subject and based on the PGC-1β sequenceinformation, determining whether the subject has a metabolic or cellularproliferation disease, disorder, or pre-disease condition or apre-disposition to a metabolic or cellular proliferation disease,disorder, or pre-disease condition, and/or recommending a particulartreatment for the disease, disorder, or pre-disease condition.

The present invention further provides in an electronic system and/or ina network, a method for determining whether a subject has a metabolic orcellular proliferation disease, disorder, or pre-disease condition or apre-disposition to a metabolic or cellular proliferation disease,disorder, or pre-disease condition wherein the method comprises thesteps of determining PGC-1β sequence information associated with thesubject, and based on the PGC-1β sequence information, determiningwhether the subject has a metabolic or cellular proliferation disease,disorder, or pre-disease condition or a pre-disposition to a metabolicor cellular proliferation disease, disorder, or pre-disease condition,and/or recommending a particular treatment for the disease, disorder orpre-disease condition. The method may further comprise the step ofreceiving phenotypic information associated with the subject and/oracquiring from a network phenotypic information associated with thesubject.

The present invention also provides in a network, a method fordetermining whether a subject has a metabolic or cellular proliferationdisease, disorder, or pre-disease condition or a pre-disposition to ametabolic or cellular proliferation disease, disorder, or pre-diseasecondition, said method comprising the steps of receiving PGC-1β sequenceinformation from the subject and/or information related thereto,receiving phenotypic information associated with the subject, acquiringinformation from the network corresponding to PGC-1β and/or a metabolicor cellular proliferation disease, disorder, or pre-disease condition,and based on one or more of the phenotypic information, the PGC-1βinformation (e.g., sequence information and/or information relatedthereto), and the acquired information, determining whether the subjecthas a metabolic or cellular proliferation disease, disorder, orpre-disease condition or a metabolic or cellular proliferation disease,disorder, or pre-disease condition. The method may further comprise thestep of recommending a particular treatment for the disease, disorder orpre-disease condition.

The present invention also provides a business method for determiningwhether a subject has a metabolic or cellular proliferation disease,disorder, or pre-disease condition or a pre-disposition to a metabolicor cellular proliferation disease, disorder, or pre-disease condition,said method comprising the steps of receiving information related toPGC-1β (e.g., sequence information and/or information related thereto),receiving phenotypic information associated with the subject, acquiringinformation from the network related to PGC-1β and/or related to ametabolic or cellular proliferation disease, disorder, or pre-diseasecondition, and based on one or more of the phenotypic information, thePGC-1β information, and the acquired information, determining whetherthe subject has a metabolic or cellular proliferation disease, disorder,or pre-disease condition r or a pre-disposition to a metabolic orcellular proliferation disease, disorder, or pre-disease condition. Themethod may further comprise the step of recommending a particulartreatment for the disease, disorder or pre-disease condition.

The invention also includes an array comprising a PGC-1β sequence of thepresent invention. The array can be used to assay expression of one ormore genes in the array. In one embodiment, the array can be used toassay gene expression in a tissue to ascertain tissue specificity ofgenes in the array. In this manner, up to about 7600 genes can besimultaneously assayed for expression, one of which can be PGC-1β. Thisallows a profile to be developed showing a battery of genes specificallyexpressed in one or more tissues.

In addition to such qualitative determination, the invention allows thequantitation of gene expression. Thus, not only tissue specificity, butalso the level of expression of a battery of genes in the tissue isascertainable. Thus, genes can be grouped on the basis of their tissueexpression per se and level of expression in that tissue. This isuseful, for example, in ascertaining the relationship of gene expressionbetween or among tissues. Thus, one tissue can be perturbed and theeffect on gene expression in a second tissue can be determined. In thiscontext, the effect of one cell type on another cell type in response toa biological stimulus can be determined. Such a determination is useful,for example, to know the effect of cell-cell interaction at the level ofgene expression. If an agent is administered therapeutically to treatone cell type but has an undesirable effect on another cell type, theinvention provides an assay to determine the molecular basis of theundesirable effect and thus provides the opportunity to co-administer acounteracting agent or otherwise treat the undesired effect. Similarly,even within a single cell type, undesirable biological effects can bedetermined at the molecular level. Thus, the effects of an agent onexpression of other than the target gene can be ascertained andcounteracted.

In another embodiment, the array can be used to monitor the time courseof expression of one or more genes in the array. This can occur invarious biological contexts, as disclosed herein, for exampledevelopment of a metabolic or cellular proliferation disease, disorder,or pre-disease condition, progression of a metabolic or cellularproliferation disease, disorder, or pre-disease condition, andprocesses, such a cellular transformation associated with the metabolicor cellular proliferation disease, disorder, or pre-disease condition.

The array is also useful for ascertaining the effect of the expressionof a gene on the expression of other genes in the same cell or indifferent cells (e.g., ascertaining the effect of PGC-1β expression onthe expression of other genes). This provides, for example, for aselection of alternate molecular targets for therapeutic intervention ifthe ultimate or downstream target cannot be regulated.

The array is also useful for ascertaining differential expressionpatterns of one or more genes in normal and abnormal cells. Thisprovides a battery of genes (e.g., including PGC-1β) that could serve asa molecular target for diagnosis or therapeutic intervention.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents, and published patent applications cited throughout thisapplication, as well as the figures and the sequence listing, areincorporated herein by reference.

EXAMPLES

Materials and Methods

All experiments were performed using the mouse PGC-1β, unless otherwisenoted.

Plasmid Construction

The full-length mouse PGC-1β cDNA clone was obtained by ligating the5′end of the PGC-1β cDNA generated by GENERACER (Invitrogen, CA) to anEST cDNA clone (GenBank Accession No. AA288169) that contained the 3′end. The presence of PGC-1β transcript in vivo was confirmed by RT-PCRusing primers located throughout the cDNA sequence. GST-PGC-1β (N350)was generated by inserting a PCR fragment coding for the N-terminal 350amino acids of SEQ ID NO:2 in frame into pGEX-5×1 vector. Fusionconstructs between GAL4-DBD and various regions of PGC-1β or HCF weregenerated by subcloning PCR-amplified cDNA fragments in frame intopCMX-Ga14 plasmid (Puigserver, P. et al. (1998) Cell 92:829-839).N-terminal Flag-tagged PGC-1β (amino acids 2-1014 of SEQ ID NO:2) wasobtained by subcloning the cDNA insert into pCATCH Flag plasmid(Georgiev, O. et al. (1996) Gene 168:165-167). All PCR fragments wereverified by sequencing.

Transient Transfection

BOSC cells and COS cells were maintained in Dulbecco's modified Eagle'smedium (DMEM) plus 10% fetal bovine serum (FBS). The cells weretransfected using FuGENE (Roche, Switzerland) according tomanufacturer's instructions. In some experiments, ligands were added tothe culture in DMEM plus 0.5% bovine serum albumin (BSA) at 50 nM for T3or 1 μM for dexamethasone 24 hours after transfection. Luciferase assayswere performed 24 hours after the addition of ligands.

RNA Expression Analysis

Total RNA was isolated from frozen mouse tissues using Trizol (Gibco,NJ). For cold exposure, C57/B16 mice were maintained at 4° C. for 6hours before sacrifice. HIB1B cells were maintained in DMEM plus 10%cosmic calf serum. To induce differentiation, confluent cells were grownin DMEM plus 10% FBS, 1 μM dexamethasone, 50 nM T3, 50 nM insulin, and0.5 mM Isobutylmethylxanthine (IBMX) for 48 hours. The differentiatingcells were then maintained in DMEM plus 10% FBS, 50 nM T3 and 50 nMinsulin. To induce UCP-1 expression, differentiated HIB1B cells weretreated with 10 μM forskolin for 6 hours before RNA isolation. For RNAanalysis, 20 μg of total RNA were separated by gel electrophoresis,transferred to a nylon membrane, and subsequently hybridized withspecific probes for various genes.

Protein Interaction Studies

Binding assays were performed as described in Puigserver et al. (1998)supra. Briefly, glutathione beads containing approximately 1 μg of GSTor GST-PGC-1α or PGC-1β fusion proteins were incubated with 5 μl of invitro translated protein for 1 hour at room temperature in 250 μlbinding buffer (20 mM HEPES, pH 7.9, 75 mM KCl, 0.1 mM EDTA, 2.5 mMMgCl₂, 0.05% NP40, 2 mM DTT, and 10% glycerol; TNT coupledtranscription/translation system, Promega, WI). Ligands were included insome binding reactions as indicated. The beads were subsequently washedfour times with binding buffer and resuspended in SDS-PAGE buffer. Thesamples were separated on a denaturing SDS gel that was dried prior toautoradiography.

For coimmunoprecipitation assays, BOSC cells were transientlytransfected with various combinations of plasmids using FuGENE. After 36hours of transfection, cells were lysed in IP buffer (100 mM Tris, pH8.0, 250 mM NaCl, 1% NP40, 1 mM EDTA, 1 mM MgCl₂, and proteaseinhibitors), and the lysate was incubated with 10 μl anti-Flag sepharosebeads (Sigma, MO) for 1 hour at room temperature. The beads were washedfour times with IP buffer and resuspended in SDS-PAGE buffer. Thesamples were then processed for immunoblotting analysis.

Example 1 Identification and Characterization of Murine and Human PGC-1βcDNAs

In this example, the identification and characterization of the genesencoding mouse and human PGC-1β is described.

Isolation of the PGC-1β cDNA

The invention is based, at least in part, on the discovery of novelgenes encoding novel proteins, referred to herein as PGC-1β. TheN-terminus of mouse PGC-1α was used to search genomic and EST databases.A partial transcript in the Celera mouse genome database (Accession No.mCT4723), which encoded a novel protein sharing a high degree ofsequence identity with PGC-1α, was designated PGC-1β. The full-lengthmouse PGC-1β cDNA sequence was obtained by ligating the 5′ end of thecDNA generated by RACE to fragments derived from an EST clone (GenBankAccession No. AA288169). The murine PGC-1β is localized to chromosome 18(FIG. 6).

The entire sequence of the 3.6 kb murine PGC-1β cDNA was determined andfound to contain an open reading frame encoding a protein of 1014 aminoacid residues. The murine cDNA sequence is set forth in FIGS. 1A-1B andin SEQ ID NO:1. The amino acid sequence of the murine PGC-1β is setforth in FIG. 2 and in SEQ ID NO:2. The coding region (open readingframe) of SEQ ID NO:1 is set forth as SEQ ID NO:3. The human PGC-1β wasidentified in the publicly available sequence database of the HumanGenome Project (GenBank Accession No. NT_(—)023152) based on homology tothe mouse PGC-1β sequence. The entire sequence of the human PGC-1β cDNAwas determined and found to contain an open reading frame encoding aprotein of 1009 amino acid residues. The human cDNA sequence is setforth in FIGS. 3A-3B and in SEQ ID NO:4. The amino acid sequence of thehuman PGC-1β is set forth in FIG. 4 and in SEQ ID NO:5.

The human PGC-1β protein of SEQ ID NO:5, which is about 70% identical tothe murine PGC-1β protein of SEQ ID NO:2, is localized to thechromosomal region 5q33 (FIG. 6). BLAST searches revealed that the PGC-1family is well conserved in other species such as chicken (GenBankAccession No. BG709977), zebrafish (GenBank Accession No. AI477804), andXenopus (GenBank Accession Nos. BI448917 and BI448253).

Analysis of the PGC-1β Molecules

Sequence analysis revealed that the mouse PGC-1β protein has similarityto PGC-1α over the entire length of the molecule, including three LXXLLmotifs at the N-terminus and one RNA recognition motif (RRM) at theC-terminus (see FIGS. 2 and 5). The human PGC-1β has a similarstructure, but has only two LXXLL motifs (see FIG. 4). The identitybetween mouse PGC-1α and mouse PGC-1β is especially high in theN-terminal activation domain (40% identical) and the C-terminalRNA-binding domain (48% identical), as shown in FIG. 5.

Sequence comparison of all PGC-1 family members, including PGC-1 RelatedCoactivator (PRC) (Andersson, U. and Scarpulla, R. C. (2001) Mol. Cell.Biol. 21(11):3738-49) revealed a novel conserved region containing atetrapeptide motif (DHDY; SEQ ID NO:7), located at about residues683-686 of SEQ ID NO:2, and at about residues 677-680 of SEQ ID NO:5,that has been previously identified in other proteins as a putativebinding site for host cell factor (HCF), a protein involved in theregulation of cell cycle progression and the assembly of a multiproteintranscriptional complex during herpes simplex virus (HSV) infection(Freiman, R. N. and Herr, W. (1997) Genes Dev. 11:3122-3127; Andersson,U. and Scarpulla, R. C. (2001) Mol. Cell. Biol. 21:3738-3749). Analysisof the PGC-1β amino acid sequence further resulted in the identificationof two glutamic/aspartic acid rich acidic domains. However unlikePGC-1α, PGC-1β lacks most of the arginine/serine rich domain (RS), aregion that has been implicated in the regulation of RNA processing(Monsalve, M et al. (2000) Mol. Cell 6:307-316).

Example 2 Analysis of Murine PGC-1β Expression Patterns

Northern hybridization analysis revealed that PGC-1β is expressed in ahighly tissue selective manner. PGC-1β mRNA is most abundantly presentin brown adipose tissue (BAT), heart and brain, all tissues notable forcontaining very high concentrations of mitochondria. These resultsstrongly suggest that PGC-1β may play an important role in mitochondrialfunction, respiration, and/or thermogenesis. Two predominant species ofPGC-1β mRNA (5 kb and 9 kb) were detected, which may be the result ofthe use of two alternative polyadenylation signals present in the 3′ endof the PGC-1β gene. Moderate levels of PGC-1β mRNA were also observed inskeletal muscle, liver, and white adipose tissue (WAT). The differencein mRNA abundance between BAT and WAT was more than 10-fold.

PGC-1α was initially identified as a transcriptional coactivator thatcontrols mitochondrial biogenesis and adaptive thermogenesis in skeletalmuscle and brown fat. In contrast to the cold-inducible expression ofPGC-1α, the expression of PGC-1β in BAT is not increased in response tocold exposure. Given its abundant expression in BAT, it was hypothesizedthat PGC-1β might be involved in the determination and/ordifferentiation of brown adipocytes. To test whether PGC-1β is regulatedduring BAT differentiation, the mouse brown fat cell line HIB1B wasinduced to undergo differentiation and examined for PGC-1β expression.Compared to undifferentiated cells, the expression of PGC-1β isupregulated on days 3 and 5 of differentiation, parallel with aconcomitant downregulation in PGC-1α expression. Upon treatment withforskolin, an activator of adenylyl cyclase, PGC-1α expression israpidly induced, along with the key uncoupling protein of brown fat,UCP1 (Klaus, S. et al. (1994) J. Cell Sci. 107:313-319). In contrast,PGC-1β expression is slightly decreased. These results indicate thatPGC-1α and PGC-1β are likely to perform distinct roles in brown fatregulation and the regulation of other brown fat functions such asadaptive thermogenesis.

PGC-1α expression is highly induced in the liver during fasting, and ithas been shown to play a direct role in the activation of hepaticgluconeogenesis in cultured primary hepatocytes and in rats (Yoon, J. C.et al. (2001) Nature 413:131-138; Herzig, S. et al. (2001) Nature413:179-183). Elevated expression of PGC-1α has also been shown inmodels of both type-1 and type-2 diabetes (Yoon et al. (2001) supra).The expression of PGC-1β is significantly increased in the liver duringfasting, a pattern that is strikingly similar to the regulation ofPGC-1α expression. These results suggest that PGC-1β may be part of theregulatory pathways that activate the hepatic adaptation during fasting,such as the elevation of gluconeogenesis, β-oxidation of fatty acids andketogenesis.

To test whether PGC-1β plays a direct role in the activation of hepaticgluconeogenesis in cultured primary hepatocytes and rats, FAO hepatomacells (FIG. 10A) or primary rat hepatocytes (FIG. 10B) were infectedwith varying doses of recombinant GFP, PGC-1α or PGC-1β viruses for 48hours. Total RNA was isolated and analyzed by Northern hybridization toexamine the expression of various genes using gene-specific probes suchas PEPCK and G6Pase for gluconeogenesis and CPT-1, MCAD and CytC forfatty acid oxidation. The results indicate that PGC-1α activates bothgluconeogenesis and fatty acid oxidation as evidenced by increasedexpression of PEPCK, G6Pase, CPT-1, MCAD and CytC, but PGC-1β onlyinduces the mitochondrial fatty acid oxidation genes CPT-1, MCAD andCytC. Thus, PGC-1β induces mitochondrial gene expression but notgluconeogenesis in hepatocytes. The inability of PGC-1β to inducegluconeogenesis, in contrast to PGC-1α, is an important distinguishingcharacteristic of PGC-1β in the arena of therapeutics for metabolicdisorders, e.g., diabetes, where gluconeogenesis-related side-effectsare undesirable.

Example 3 Interaction of Murine PGC-1β with Nuclear Receptors

PPARα and HNF4α are important transcription regulators implicated inhepatic fatty acid oxidation and gluconeogenesis during periods of fooddeprivation (Kersten, S. et al. (1999) J. Clin. Invest. 103:1489-1498).It was therefore determined whether PGC-1β, like PGC-1α, couldphysically interact with and coactivate these transcription factors. TheN-terminus of PGC-1β (amino acid residues 1-350 of SEQ ID NO:2), whichcontains three putative nuclear receptor binding motifs (LXXLL motifs)is able to “pulldown” in vitro translated HNF4α and PPARα with similarefficiency as PGC-1α. Furthermore, the full-length PGC-1α is readilycoimmunoprecipitated with HNF4α when coexpressed in BOSC cells,indicating in vivo association of these two proteins. The interactionbetween PGC-1β and PPARα is slightly increased in the presence ofWy-14643, a PPARα ligand. In contrast, the recruitment of PGC-1β by RARα(retinoic acid receptor α) and TRβ (thyroid hormone receptor β) ishighly dependent on their respective ligands. Consistent with apotential role in regulating hepatic gluconeogenesis, PGC-1β potentlyenhances the transcriptional activity of GR (glucocorticoid receptor)and HNF4α on reporter constructs containing multimerized cognate bindingsites (FIGS. 7A and 7B, respectively). Coactivation of GR by PGC-1β isstrictly dependent on the presence of dexamethasone, a syntheticglucocorticoid, indicating ligand-dependent recruitment of thecoactivator to the receptor; the latter is also observed for TRβ andRARα (FIG. 7D). In addition to coactivating NRs, PGC-1β is also a potentregulator of the transcriptional activity of NRF1, a centraltranscription factor in the control of mitochondrial biogenesis (FIG.7C). These results demonstrate that PGC-1β regulates the transcriptionalactivity of an array of nuclear receptors and other transcriptionfactors through direct physical association with these factors.Significantly, these include factors known to be important inmitochondrial biogenesis, fatty acid oxidation, and gluconeogenesis.

Example 4 Analysis of PGC-1β Transcriptional Activity

This example examines whether PGC-1β has autonomous transcriptionalactivity when fused to a heterologous DNA binding domain. PGC-1βpotently activates transcription from a UAS-luciferase reporterconstruct when fused to the DNA binding domain of yeast GAL4 (FIG. 8).Deletion of the C-terminus further increases its transcriptionalactivity. The transactivation domain of PGC-1β is localized to theN-terminus of the protein.

Sequence alignment of all PGC-1 family members revealed severalconserved patches of amino acids that may be important for theirfunction. One such region contains a tetrapeptide motif, DHDY (SEQ IDNO:7), that is a putative HCF-binding motif (HBM) with a consensussequence of [D/E]-H-X-Y, wherein [D/E] indicates either D or E, and Xindicates any amino acid residue. Proteins containing this motif havebeen shown to associate with HCF, including HSV viral protein VP16 and abasic leucine-zipper protein, LZIP (Freiman, R. N. and Herr, W. (1997)Genes Dev. 11:3122-3127). HCF does not bind DNA by itself; however, itcan be recruited by DNA-binding transcription factors such as Oct-1 andfunctions as a scaffold for the assembly of transactivation complexes(Vogel, J. L. and Kristie, T. M. (2000) EMBO J. 19:683-690).

PGC-1α and PGC-1β may mediate the recruitment of HCF-containing proteincomplexes to the NR binding sites, thereby modulating NR-regulatedtranscription. To test whether PGC-1α and PGC-1β are physicallyassociated with HCF in vivo, Flag-tagged HCF was cotransfected withGAL-PGC-1α or PGC-1β and analyzed by immunoprecipitation followed bywestern blot analysis. HCF strongly interacts with both PGC-1α andPGC-1β, thus identifying a novel interaction partner for PGC-1-relatedcoactivators. Recruitment of HCF to PGC-1β increases the transcriptionalactivity of PGC-1β three fold in a cotransfection assay (FIG. 9A). Onthe other hand, PGC-1β can be recruited by the N-terminal 380 aminoacids of HCF, a domain that has been shown to interact withHBM-containing proteins, and potently increases the transcriptionalactivity of HCF fused to the GAL4 DBD (FIG. 9B). Coactivation of HCF wasalso observed for PGC-1α. These results demonstrate that HCF canfunction in complex with PGC-1α or PGC-1β to activate transcription fromtarget genes.

Example 5 Expression of Recombinant PGC-1β Protein in Bacterial Cells

In this example, PGC-1β is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, PGC-1βis fused to GST and this fusion polypeptide is expressed in E. coli,e.g., strain PEB199. Expression of the GST-PGC-1β fusion protein inPEB199 is induced with IPTG. The recombinant fusion polypeptide ispurified from crude bacterial lysates of the induced PEB 199 strain byaffinity chromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 6 Expression of Recombinant PGC-1β Protein in Mammalian Cells

To express the PGC-1β gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire PGC-1β protein and a FLAG tag fused in-frame to its 5′ end of thefragment is cloned into the polylinker region of the vector, therebyplacing the expression of the recombinant protein under the control ofthe CMV promoter.

To construct the plasmid, the PGC-1β DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the PGC-1βcoding sequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, and the 3′ untranslated region ofthe PGC-1β cDNA. The PCR amplified fragment and the pCDNA/Amp vector aredigested with the appropriate restriction enzymes and the vector isdephosphorylated using the CIAP enzyme (New England Biolabs, Beverly,Mass.). Preferably the two restriction sites chosen are different sothat the PGC-1β gene is inserted in the correct orientation. Theligation mixture is transformed into E. coli cells (strains HB101, DH5α,SURE, available from Stratagene Cloning Systems, La Jolla, Calif., canbe used), the transformed culture is plated on ampicillin media plates,and resistant colonies are selected. Plasmid DNA is isolated fromtransformants and examined by restriction analysis for the presence ofthe correct fragment.

Mammalian cells are subsequently transfected with the PGC-1β-pcDNA/Ampplasmid DNA using the calcium phosphate or calcium chlorideco-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the PGC-1β polypeptide is detected byradiolabeling (³⁵S-methionine or ³⁵S-cysteine available from NEN,Boston, Mass., can be used) and immunoprecipitation (Harlow, E. andLane, D. Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988) using an FLAG specific monoclonalantibody. Briefly, the cells are labeled for 8 hours with ³⁵S-methionine(or ³⁵S-cysteine). The cells are lysed using detergents (RIPA buffer,150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5).andprecipitated with an HA-specific monoclonal antibody. Precipitatedpolypeptides are then analyzed by SDS-PAGE using a PGC-1β specificmonoclonal antibody.

Example 7 Role of PGC-1 in Cellular Defense

To test whether PGC-1β induces mitochondrial biogenesis in murinemyotubes, C2C12 myotubes were infected with recombinant adenoviruses.Total RNA (FIG. 11A) or DNA (FIG. 11B) was isolated and examined forgene expression or mitochondrial content, respectively. The resultsshowed that both PGC-1α and PGC-1β activate mitochondrial geneexpression.

Expression of enzymes involved in free-radical metabolism such assuperoxide dismutase (Mn-SOD) and glutathione peroxidase (GPx) inresponse to PGC-1α and PGC-1β was also tested (FIG. 11A). The resultsshow that enzymes involved in free radical metabolism are highlyelevated in response to both PGC-1α and PGC-1β. Thus, the PGC-1 familyof coactivators play an important role in the cellular defense againstfree radical damage.

Example 8 Expression of PGC-1β Protein in Neuroblastoma Cells

To test whether PGC-1β induces mitochondrial gene expression inneuroblastoma cells, human neuroblastoma cells were cultured andinfected with recombinant adenoviruses expressing PGC-1α and PGC-1β,respectively. Total RNA was isolated and analyzed by hybridization usingspecific probes. The results show that both PGC-1α and PGC-1βsignificantly increase mitochondrial gene expression. The ability ofPGC-1 coactivators to regulate mitochondrial gene expression in neuronalcells indicates that PGC-1 may be an important regulator of brain energymetabolism. Since abnormal mitochondrial function is usually implicatedin neurological disorders such as Parkinson's disease, PGC-1 may be animportant therapeutic target in the arena of neurodegenerative diseases.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated polypeptide comprising an amino acid sequence which is atleast 97% identical to the amino acid sequence of SEQ ID NO: 5, whereinthe polypeptide modulates one or more of the following biologicalactivities: activity of a nuclear receptor and/or nuclear respiratoryfactor 1 (NRF1); brown adipose cell determination and/ordifferentiation; mitochondrial activity and/or biogenesis; or fatty acidβ-oxidation.
 2. The isolated polypeptide of claim 1 comprising the aminoacid sequence of SEQ ID NO:
 5. 3. The polypeptide of claim 1, furthercomprising a heterologous amino acid sequence.
 4. An isolatedpolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence which is at least 97% identical to the nucleic acidsequence of SEQ ID NO: 4 or 6, wherein the polypeptide modulates one ormore of the following biological activities: activity of a nuclearreceptor and/or nuclear respiratory factor 1 (NRF1); brown adipose celldetermination and/or differentiation; mitochondrial activity and/orbiogenesis; or fatty acid β-oxidation.
 5. The isolated polypeptide ofclaim 4, further comprising a heterologous amino acid sequence.